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  • Published: 24 March 2023

LNA blockers for improved amplification selectivity

  • Jaime Prout 1 ,
  • Michael Tian 1 ,
  • Alicia Palladino 1 ,
  • Jason Wright 1 &
  • John F. Thompson 1  

Scientific Reports volume  13 , Article number:  4858 ( 2023 ) Cite this article

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  • Molecular biology

LNA-containing oligonucleotides bind DNA more tightly than standard DNA, so they can interact with targeted sequences and affect multiple processes. When a desired DNA is present at low concentrations relative to nearly identical undesired DNAs, LNAs can block amplification of unwanted DNAs. Using a short rAAV and synthetic DNA sequence as a model, we studied the length, number, and positioning of LNA bases to improve blocker effectiveness. Oligonucleotides 18–24 bases long with LNAs at every other position were most effective. Highly degenerate targets were used to characterize the impact of mismatches on blocking. Mismatches at LNA ends had little impact on blocking activity. Single and double mismatches were tolerated with longer blockers, especially if the mismatches were near LNA ends. Shorter LNAs were more selective, with > 1 mismatch preventing effective blocking. Neither the strand to which a blocker bound nor the distance between the blocker and priming sites greatly impacted blocking efficiency. We used these findings to design blockers of wild-type DNA versus the single-base A1AT PiZ allele. Blockers are most specific when the mismatch is located away from the LNA 5′ end. Pairs of partially overlapping blockers on opposite strands with a centrally-located mismatch have maximal activity and specificity.

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Introduction

Locked Nucleic Acid (LNA) nucleotides are identical to natural nucleotides except for a methylene bridge spanning the deoxyribose sugar 1 , 2 which makes them more stable in double-stranded structures and more resistant to degradation 3 . The higher melting temperatures (T m s) of oligonucleotides that include LNA bases provide greater specificity and new functions. They have been used successfully as PCR primers/probes 4 , 5 , 6 , as antisense reagents 7 , 8 , 9 , as selective binders for distinguishing single-nucleotide variants 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , as agents for selective capture/degradation 19 , 20 , and as polymerization/splicing blockers 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 . Each of these roles requires that the LNA bind tighter than the corresponding pure DNA, but some functions may also require additional attributes that could be affected by the number and location of LNA bases within the oligonucleotide. Furthermore, proteins may interact with LNAs differently than with standard DNAs, dictating whether LNA or DNA should occupy a particular position.

While LNA blockers have been used in multiple situations, those studies have little commonality to provide insight into preferred designs. Some use chimeric 11 , 12 , 13 , 15 , 21 , 23 , 26 or pure 10 , 17 , 27 LNAs of 16 nt or shorter, while others use chimeric LNAs of 20 nt or longer 16 , 22 , 24 For 20-mers, there are more than one million possible LNA-DNA configurations for each of the more than 1 trillion possible sequences. In addition, functional predictions of how different LNAs will perform as primers, blockers, or in other roles are even less well characterized because additional factors beyond the well-studied T m 29 , 30 , 31 , 32 , 33 may play a critical role in how different LNAs perform 34 , 35 , 36 .

Blocking with LNAs is not the only method that has been used to prevent the amplification of majority DNAs when the detection of minority DNAs is desired. Other nucleic acid analogs like Peptide Nucleic Acids (PNA) selectively block amplification 37 , 38 . Choice of PNAs versus LNAs for a particular application will depend on the details of the system. LNAs have the advantages of lower cost and the ability to make longer molecules to reduce the number of perfect binding sites in complex genomes. PNAs can bind more strongly and are not susceptible to nucleases. A concern for PNAs can be specificity where the shorter PNAs may bind multiple genomic sites and potentially interfere with other DNAs of interest. In our case, we often wish to amplify DNAs of several kilobases or more arising from multiple genomic regions. Knowing that blockers will not interfere with DNAs elsewhere in the genome can be important. Thus, the more predictable and selective LNAs are preferred in our application though PNAs can be suitable in other situations.

One method for classifying blockers is whether they prevent the annealing of a PCR primer or prevent the elongation of that primer 39 . Annealing blockers are often used when the priming site for the desired amplicon is different by only one or by a small number of bases relative to the undesired amplicon. When few differences are present, shorter LNAs are often used to enhance the difference between perfect and imperfect binding. When the regions available for priming of the desired and unwanted DNAs are identical, annealing blockers cannot be used. Instead, sequences specific to the interior of the undesired amplicon must be bound by an elongation blocker. Our aim was to preferentially amplify integrated recombinant Adeno-Associated Virus (rAAV) while preventing the amplification of episomal copies of the transduced rAAV. Episomal DNA contains the same sequences as integrated DNA 40 , so elongation rather than annealing blockers must be used. Because other rAAVs and DNAs of interest may contain different sequences and hence need different amplification conditions, it is helpful to understand how to design effective LNA elongation blockers for different contexts and applications.

The process of blocking amplification involves many individual components. As diagrammed in Fig.  1 , the priming and blocking oligonucleotides and the target DNA could all potentially participate in unimolecular, bimolecular, and higher-order interactions. During primer/blocker design, sequences are picked that minimize internal secondary structure and the formation of homo/heterodimers. The amplifying polymerase could bind to both oligo/target complexes and either stabilize or destabilize those interactions, with or without the additional complexity of polymerization. In addition to these potential interactions, there is also the likelihood that any component could interact with other DNAs in the mix because the reason for wanting to block the target DNA is to improve the signal relative to other related DNAs present in the complex mixture.

figure 1

Potential interactions among intended reactants during PCR blockage. Potential intra- and inter-molecular interactions are shown for the PCR primer (black), the LNA blocker (red), the target DNA (blue), and the DNA polymerase (green). The DNAs/LNA can undergo folding into interfering secondary structures (1). The primer/blocker can potentially interact in a non-productive complex (2) with each other or in a productive or non-productive complex with the target DNA (3). LNAs have a slower off-rate versus DNAs 46 . The DNA polymerase can then bind the primer/blocker/target complex (4) and either extend the primer to generate an Amplified Target (5) or, if polymerization is halted by the LNA when the LNA is bound to the target DNA, a Blocked Target (6).

The large number of potential substrates and conditions prevent a complete analysis of all LNA lengths and sequences. For these experiments, we have restricted the total length of the LNA/DNA chimeras to those long enough to bind and provide reasonable specificity within the human genome (≥ 16 nt) while not so long as to allow binding to occur despite the presence of multiple mismatches. For the initial experiments (Table 1 ), the target DNA was a plasmid containing a sequence derived from a multiple cloning site and episomal rAAV 40 . Subsequent experiments (Table 2 ) used a synthetic 175mer DNA (Template_0) with the same targeted sequence flanked by different priming sequences (Fig.  2 ). Additional experiments used other DNA targets as described in Table S1 . Generally, we used DNA polymerase GXL that lacks 5′ > 3′ exonuclease activity. All LNA-containing oligonucleotides were capped at both the 5′ and 3′ ends to minimize degradation and prevent extension as a primer. Initially, some LNAs were made with phosphorothioate linkages to prevent degradation. No effect was seen (data not shown), so later LNAs did not include such linkages.

figure 2

Template_0 target DNA and primer/LNA binding locations. The target oligonucleotide to be blocked (Template_0) includes a left primer region (1–43), a multiple cloning site (MCS) with SbfI, XbaI, and SnaBI sites (44–59), AAV2 sequence (4495–4558 in http://www.ncbi.nlm.nih.gov/nuccore/AF043303.1 , 60–124, shown in red) and a right primer region (125–175). Two forward primer sequences on the left, F1 and F3, and three reverse primers on the right, R1, R3A, and R3B (blue arrows) were used to amplify the sequence with or without LNA blockers. Each LNA blocker (green double arrows) was made in four versions, two corresponding to the top strand (F, forward) and two corresponding to the bottom strand (R, reverse). The pair of F LNAs and the pair of R LNAs were synthesized starting with LNAs at either position 1 or position 2. These are listed individually in Table 2 and Table S1 .

Qualitative assays were run at varying LNA concentrations to identify where to focus with length and the number/positioning of LNAs (Table 1 ). For these studies, varying numbers of LNAs were situated in the middle, at either end, or throughout the oligonucleotide. The LNAs were added to PCR reactions at 0.1 µM or 10 µM, and all were targeted to the same DNA site for consistent comparisons. We found that maximum effectiveness occurred when chimeric molecules were ≥ 20 nt and half of the positions were substituted with LNAs. Furthermore, an even distribution of LNAs throughout the molecule (LNA_10) was more effective than LNAs clustered together (3′MCS, 3′MCS_5′, and 3′MCS_3′), so all later LNAs contained alternating LNA/DNA.

Based on the results in Table 1 , a new set of 18–30 nt blockers was synthesized with alternating DNA/LNA nucleotides (Fig.  2 , Table 2 , and Table S1 ). LNAs were made with sequences identical to the top (forward, F) strand and the bottom (reverse, R) strand of the 175 nt synthetic Template_0 (Fig.  2 ). In most cases, pairs of oligos were made, varying with respect to whether the first LNA was placed at position 1 or position 2. Oligonucleotides with names ending in 1 started with the first nucleotide as LNA, while oligonucleotides with names ending in 2 started with the second nucleotide as LNA with DNA/LNA alternating thereafter. All chimeras with LNAs starting with the second position had higher predicted T m s than the equivalent molecules starting with LNA in the first position (Table S1 ). Individual LNAs were used to block amplification at 1 µM, with their effectiveness measured using quantitative capillary electrophoresis relative to samples with no LNA added (Fig. S1 , Table 2 ). Effectiveness varied significantly among oligonucleotides. In most cases (10 of 12 tested), blockers with LNAs starting in the second/even positions were more effective than the same blockers with LNAs in the first/odd positions. Nearly half (15 of 34) of the blockers tested with primers F1/R1 yielded > 90% blocking, and another 6 blocked > 80%.

The initial results with the F1/R1 primer pair suggested that blockers separated on the same strand from the primers by 20–40 nt did not work as well as those positioned further away. The length of DNA gaps between the primers and blockers was changed to determine whether this affected blocking efficiency. Primer R1 was replaced with primers R3A and R3B that were 61 and 43 nt closer to the blocker binding sites to generate altered primer/blocker spacing. Blocker effectiveness did not change when the new primers were 20–40 nt away from the blocker, suggesting that, if there is a gap distance effect, it is not strong. Similarly, moving the primer by 4 bp to change helical orientation on the forward strand (primer F1 versus F3) had minimal effect on blocking (Table 2 ).

While the blocking efficiency of LNAs is a critical feature, some applications require specificity so that non-targeted sequences are minimally affected. To assess how target mismatches affect blocking efficiency, Template_D was synthesized with a blocker binding region containing multiple partially degenerate positions located at the site bound by blockers 18_F2, 18_R2, 20_F2, and 20_R2 (Fig. S2 ). Template_D had a different sequence relative to Template_0 beyond the 20 nt identical region bound by these blockers (discussed in Fig. S2 legend). The degeneracy introduced by using a mixture of nucleotides during synthesis allows the creation of a highly complex pool of DNA molecules with a wide range of variants that can be used as a substrate for determining the effect of DNA mismatches on LNA blocking. This pool contained potentially over 1 trillion different DNA molecules with randomly situated variants within the degenerate region. Because the synthetic nucleotide pool favored the reference sequence (79%), molecules averaged only 4 variants each. In addition to the 18- and 20-mers that could be tested on both Template_0 and Template_D, we also wanted to test Template_D with a longer blocker. A new pair of 24 nt LNAs was made (24D_F2 and 24D_R2). Even though the 24D blockers were 24 nt long, they covered only 21 degenerate positions because three positions bound by 24D consist of 100% reference sequence.

When there is a constant level of degeneracy in DNA, the frequency of variants within a given length can be predicted mathematically using classic combination/permutation equations ( https://en.wikipedia.org/wiki/Combination ). When R is the proportion of correct reference sequence and L is the length of the degeneracy, the frequency of a perfect reference sequence (no variation) is \({R}^{L}\) . For DNA synthesized to be 79% Reference/21% Mismatch, the percent of reads matching the reference sequence perfectly is predicted to be 1.4% for an 18mer and falls to 0.7% for a 21mer. For predicting the number of variant sites in all molecules, the expected frequency for any given number of mismatches (MM) in the target region of length L can be calculated from the equation:

For example, to determine the frequency of molecules with 3 mismatches in a 20mer that was 79% reference sequence, one would calculate 100 * (0.79) 17 * (0.21) 3 * (20!)/((3!) * (17!)) yielding 19.2%, meaning that 19.2% of all 20mers should have exactly three mismatches somewhere within their sequence. The predicted read frequencies as a function of the number of mismatches for 18, 20, and 21 degeneracies are shown in Fig.  3 A. The frequencies for the 24D LNAs were calculated with 21 degeneracies because three template positions bound by the LNAs were reference, not degenerate. To compare the predicted frequency with the actual frequency, the target DNA pool was amplified with primers that bound the constant primer region outside of the degenerate regions. After barcoding, the amplified samples were sequenced using a MiSeq and the frequency of variant positions compared to the predicted results. The mismatch frequency for the samples with no LNA blocking closely mirrors the calculated values for 79% reference/21% mismatch (Fig.  3 A and Table S2 ). Values were calculated only up to 10 mismatches because, with these synthetic parameters, more than 99% of DNA molecules have 10 variants or fewer.

figure 3

Predicted and observed mismatch read frequencies. ( A ) The predicted mismatch frequency (solid fill) and observed variant read frequency for unblocked samples (hatched fill) as a percent of total reads normalized by sample for 0–10 variants is shown for degenerate DNA lengths of 18 (blue), 20 (gray), and 21 (red) nt. These predictions were made using the combination equation in the text where R = 0.79, L = 18 or 20 or 21 and MM = 0–10. > 99% of all molecules with these degeneracy lengths and 79% reference sequence should have 10 or fewer variants so higher variant values are not shown. Even though the 24D blockers are 24 nt long, a length of 21 is used for its calculations because only 21 of the 24 positions on the target DNA are degenerate. The calculated frequencies are nearly identical to the observed variant frequencies in DNA reads when the amplification is not blocked by LNAs (Table S2 ). ( B ) For forward (F2) or reverse (R2) blockers covering 18, 20, or 21 degenerate positions, the percent of reads for each sample versus no LNA present is shown for each mismatch value. The number of mismatches allowed while still retaining blocking activity varies by length.

To determine the impact of mismatches on blocking, amplifications of the complex pool of DNA sequences that make up Template_D were performed with either no LNA or individual LNAs at 1 µM. The primers used to amplify Template_D each bound identical, non-degenerate regions of the DNAs while the blockers had trillions of different sequences to which they could bind. The ability of LNAs to bind certain sequences caused blocking of those DNAs while others amplified normally. If variants in the target pool are differentially affected by LNA blockers, the read distribution should change accordingly. When blocked samples were examined (Fig.  3 B), all 18 and 20 nt blockers had 27–36% as many reads with no mismatches as the unblocked samples, while the 24 nt blockers had only 8.4–8.6% as many reads with no mismatches as unblocked samples, showing that specific blocking had occurred, and the amount of blocking was length dependent. The read frequency for each mismatch was very similar when 18_F2/18_R2 and 20_F2/20_R2 were compared, indicating that the strand being blocked does not matter with this target. There is a greater deviation between 24D_F2 and 24D_R2, but this may be due to the asymmetric location of degenerate positions in the two blockers. 24D_R2 has three fixed target positions near its 5′ end, while the fixed positions for 24D_F2 are near its 3′ end.

For the 18 and 20 nt blockers, there were 54–77% as many reads with 1 mismatch relative to no LNA. There is only a slight difference in reads (85–96% as many reads relative to no LNA) with 2 mismatches relative to no LNA, indicating that any mismatches lead to poor blocking at these LNA lengths. All blocking activity was lost with three or more mismatches for 18 and 20 nt blockers. With the 24 nt blockers, there was significant blocking with 0, 1, and 2 mismatches. There was lower but measurable blocking (59–86% as many reads relative to no LNA) with 3 mismatches. This indicates that the specificity of blocking is dependent on the length of the blocker and longer LNAs are capable of blocking to some degree, even in the presence of up to three suitably placed mismatches.

To assess the relative importance of different positions within the LNAs for blocking specificity, the locations of mismatches within all reads with a single mismatch were examined. The positional frequency of single mismatches in the No LNA sample relative to all reads in the sample is uniform as each mismatch has no effect on amplification (Table S2 ). The frequency of single mismatch reads by position relative to all reads in 18 and 20 nt blocked samples varies significantly and is shown in Fig.  4 A. The frequencies are given as a percentage relative to the values observed in the unblocked sample. Because effective blocking was observed with both one and two mismatches with 24D_F2 and 24D_R2, the positional effects for both one and two mismatches are shown for those blockers (Fig.  4 B). If all positions are equally important for blocking, all blocked samples would have the same percentage across the length of the LNA. Indeed, that is the observation with the 24D_F2 LNA with a single mismatch: it does not matter where the mismatch occurs, there is 90–95% blocking, independent of position. For all other conditions with one or two mismatches, there is a positional dependence on blocker effectiveness. Positions very close to either end of the blocked region have lower relative read frequencies than the more centrally located positions. The lower read frequency for mismatches near the blocker ends means templates with those mismatches are blocked despite the mismatch, so they are less represented among all reads. If selectivity of blocking is desired, significant blocking in the presence of mismatches is not a good thing. Though the position of the mismatch matters, the identity of the base substitution at a given position is generally irrelevant. At most positions, all mismatches have similar blocking effects (Table S3 ). There are some differences but no obvious trends.

figure 4

Blocking as function of position. ( A ) The relative read frequency for amplification with blockers versus amplification with no LNAs is shown as a function of position. In panel A, the frequency of reads with single mismatches is shown for 18 (red) and 20 (blue) nt LNA blockers. Blockers identical to the forward strand are shown with solid lines/symbols, and blockers identical to the reverse strand are shown with dashed lines/open symbols. The overall shape for all forward primers and for all reverse primers is similar. ( B ) LNAs blockers that cover 21 degenerate positions are shown with data for reads for one mismatch (1MM, solid lines/symbols) and for reads with two mismatches (2MM, dashed lines/open symbols). Blockers identical to the forward strand are shown in blue, and the reverse strand in red. Blocking in the presence of mismatches is not necessarily desirable as it indicates a lack of selectivity.

An additional common feature among blockers that did not perform well was the presence of an A on the same strand adjacent to the 5′ end of the blocker binding site. When the base was an A, the average blocking effectiveness was 42% (n = 5), while it was 84% for C (n = 16), 93% for G (n = 4), and 81% for T (n = 9). To test the same blockers with a different adjacent sequence, two new templates were made (Template_A1 and Template_A2, Figs. S3 and S4 ). Many LNA blockers shown in Table 2 had overlapping binding sites, so making a base change adjacent to one binding site created a mismatch within other binding sites. Only the subset of the original blockers that maintained perfect binding sites were tested on the new templates. For the blockers tested, all preceding non-As were changed to A, and all preceding As were changed to another base (Table 3 , Figs. S3 and S4 ). The eleven changes from C/G/T to A resulted in an average decrease in efficiency of 21% while the three changes from A to G/T resulted in an average increase in efficiency of 15%. In addition to Template_A1 and Template_A2 which have uniformly altered sequences, it is also possible to use Template_D to address the question of adjacent sequence. Two of the blockers used, 18_R2 and 24D_R2, have a degenerate position adjacent to the 5′ end of their binding site on that target DNA. Thus, the frequency of each base prior to their binding sites can be examined for differences. There do not appear to be significant base-specific effects at those sites with Template_D; so, if adjacent bases have an impact, the effect is small, may be more complex than just a single base, or may be evident only in certain conditions.

A key component of the overall amplification and blocking process is the DNA polymerase. The experiments described thus far were carried out with GXL polymerase, which has 3′ > 5′ exonuclease activity, but no 5′ > 3′ exonuclease activity as some other polymerases have. Two additional polymerases were tested in parallel against a subset of LNA blockers carried out in conditions and temperatures recommended by the suppliers. As shown in Table 4 , the two polymerases which lack 5′ > 3′ exonuclease activity, GXL and Q5, had similar blocking profiles. The polymerase that does exhibit 5′ > 3′ exonuclease activity, GoTaq, behaves differently. Template_U has no LNA binding sites, but it is blocked to the same extent as Template_O with 6 of the 8 blockers tested. There may be a small amount of specific blocking with the two best blockers, 30_R1 and 30_R2. Literature results for blocking of Taq have produced discrepant results 11 , 41 . Those experiments were run at different temperatures, so we also tested GoTaq with an extension temperature of 60 °C where LNA blocking was reported. We find specific blocking at that temperature though not as much as found with the other polymerases.

To see if these design properties could be extended to another target of biological interest, LNA chimeras were designed to the wild-type allele of A1AT to see whether its amplification could be blocked specifically relative to the single base PiZ mutation which is linked to COPD and other respiratory diseases 42 . Four pairs of LNAs matching the wild-type sequence were made (Fig.  5 A). These LNA pairs placed the base corresponding to the PiZ mutation at the 5′ end and at the third, fifth, or seventh position in from the 5′ end. This was done for both DNA strands (Forward and Reverse). All LNAs started at the second position and alternated through the molecule. Five of the eight were 16 nt long but the three with the lowest T m s were extended one additional base to make the T m s more similar (Table S1 ). The region around the PiZ allele was amplified from human genomic DNA using primers listed in Table S1 to generate a 646 bp product. LNAs were titrated versus wild-type DNA to find concentrations of each that would yield extensive but not complete blocking. These concentrations varied significantly and were independent of T m . The best A1AT LNA blocker, A1AT_R1_2, was 30 times more potent than the worst blocker, A1AT_F1_2. Based on this, genomic DNA with either wild-type or PiZ alleles was amplified with the eight individual LNAs as well as with each pair of corresponding F/R LNAs. As shown in Fig.  5 , concentrations that yielded significant blocking of wild-type A1AT had no effect on PiZ A1AT except for A1AT_F1-2 and A1AT_R1_2 which blocked the PiZ DNA nearly as well as wild-type despite the mismatch at the 5′ end for both. The combination pairs all blocked wild-type DNA better than the individual blockers. When LNA concentrations were increased 16-fold, five LNAs blocked PiZ DNA amplification less than 30% (A1AT_F3-2, A1AT_F5-2, A1AT_R3_2, A1AT_R5_2, and A1AT_R7_2) while A1AT_F1-2 and A1AT_R1_2 blocked it more than 95%. More effective blocking of PiZ DNA was also observed with all combinations except for A1AT_F7_2/ A1AT_R7_2 which was similar to the individual blockers. This blocker pair overlaps each other by 13 nt so it is likely they will bind each other in solution, potentially affecting their ability to bind to both wild-type and PiZ DNA.

figure 5

Blocking A1AT . ( A ) The sequence surrounding the PiZ allele in A1AT is shown (central base, R/Y). G/C is the wild-type sequence and A/T is the PiZ allele. LNAs are named with an F if identical to the top strand and R if identical to the bottom strand. The number adjacent to F/R indicates where the PiZ allele is in the LNA. Individual LNAs and how they overlap are shaded. Because each LNA varies by two in length, the alternating LNAs are positioned identically in these series. Based on potency in initial studies at 1 and 10 µM, concentrations were titrated to obtain good but not complete blocking at the lowest concentrations used. The lowest concentration for A1AT_R1-2 was 0.1 µM, followed by A1AT_F3-2, A1AT_R3-2, and A1AT_R5-2 at 0.2 µM, A1AT_F5-2 and A1AT_R7-2 at 0.5 µM, A1AT_F7-2 at 1 µM, and A1AT_F1-2 at 3.3 µM. ( B ) Blocking of wild-type DNA with LNA concentrations listed in ( A ). ( C ) Blocking of PiZ DNA with LNA concentrations listed in ( A ). ( D ) Blocking of PiZ DNA with LNA concentrations 16-fold higher than listed in ( A ). Data is not shown for the 16 × wild-type DNA because all LNAs completely blocked amplification.

PCR has been used to routinely amplify rare DNA sequences from complex mixtures. When the desired DNA has unique sequence characteristics, high degrees of amplification are readily achieved. When the targeted sequence is overwhelmed by an excess of highly similar sequences, selective amplification may not be as straightforward, and detecting specific rare DNAs in a mix of nearly identical molecules can be challenging. For example, when seeking to identify rAAV DNAs that have integrated into the host cell genome, there can be a high level of episomal DNA, up to thousands of copies per cell, obscuring less frequent genomic integration events. Sherman et al. 24 used 27–32 nt blockers with 9 LNAs to address a similar issue with retroviral and lentiviral genomic integrations. These viral integration studies encountered a less severe problem due to the absence of high copy episomal DNA. Similar scenarios, such as complex metagenomic samples, somatic mutations, multiple paralogs, repetitive sequences, or pseudogenes, can also be complicated by DNA ratios for which the desired amplification is challenging. In the rAAV scenario, unique sequences are present in the non-integrated segments of the episomal virus relative to the integrated version, providing wide latitude in sequence choice. In other situations, there may be only one or a small number of changes, limiting the flexibility in blocker choice.

Based on thermodynamic studies 29 , 30 , 31 , 32 , 33 , all the oligonucleotides with LNA substitutions we studied should stably bind the target DNAs of interest. While LNA binding is a minimal requirement, the positioning and number of LNAs may or may not affect function, so relying on binding alone to predict function would be ill-advised. A 50% fractional LNA content performed best in initial experiments, especially when LNAs were located throughout the blocker rather than clustered in one region. Our initial observations suggesting that alternating LNA substitutions are functionally superior is consistent with other studies that indicate special LNA binding properties with such an arrangement 43 , 44 . Blocking activity was generally better when the LNAs started at the second position rather than the first.

To allow analysis of trillions of different targets, a highly degenerate pool of DNAs was synthesized for blocking studies. For DNA with 18–21 degenerate positions with the potential for all four bases at each position, this was equivalent to testing up to 4 trillion different targets simultaneously. Because the target DNA was synthesized to favor the reference sequence, molecules averaged four variants each, but could have anywhere from 0 to 21 variant positions. It is unlikely that most molecules with > 10 variants are represented because the pool was designed to test DNAs with fewer variants. We found that the exact sequence of mismatches did not generally have an effect at any blocked position. However, the position of the mismatch on the template does matter. Reads with mismatches very close to either end of the blocker were found far less often than more centrally located mismatches, indicating that mismatches at the ends still allowed less specific blocking, while more interior mismatches prevented blocking. If blocking of both perfect and slightly imperfect matches is not an issue, this positional effect will not matter.

However, when the intent is to distinguish between two DNA sequences that differ by only a single base, the positional effect is critical. Placing the mismatch away from the blocker ends is necessary if there is to be good blocking with a perfect match and no blocking with a single mismatch. We used the PiZ single base mutation in the A1AT gene as an example for evaluating the generalizability of these findings. Four pairs of LNAs that placed the mismatch position at the 5′ end or 3, 5, or 7 nt into the LNA confirmed that internal LNA positions are superior to the 5′ end with respect to specificity. While all LNAs block the wild-type allele, the 5′ end mismatch also blocks PiZ allele DNA with nearly the same effectiveness. When LNAs binding to both strands are used, improved blocking is observed. Of the LNAs we tested, the combination of A1AT_F7-2 and A1AT_R7-2 had the greatest overlap between them, 13 nt. Even with this high degree of overlap that could lead to stable binding to each other, blocking of the targeted allele remained strong though with slightly reduced specificity toward the mutation. The most effective and specific blockers had the mismatch located 3 or 5 nt from the 5′ end.

Previously, there have been reports that polymerases with 5′ > 3′ exonuclease activity eliminate LNA blocking activity 11 , while others have found no effect 41 . In our hands, Taq polymerase with 5′ > 3′ exonuclease activity is blocked non-specifically by LNAs when amplification is carried out at the recommended extension temperature. Amplification is inhibited 26–53% (Table 4 ) when there are specific target binding sites present (Template_0) and 32–48% when there are no LNA binding sites present (Template_U). There may be some specific blocking with the best blockers, 30_R1 and 30R_2. GXL and Q5 polymerases, which both lack 5′ > 3′ exonuclease activity, are non-specifically inhibited to a lesser degree and have much higher specific inhibition when there is a target binding site. If the amplification conditions that produced disparate results in the literature are compared, the most notable difference is the extension temperature during amplification. When primers are extended at 72 °C 11 , polymerases with 5′ > 3′ exo activity exhibit no blocking while extension at 60 °C 41 retains blocking. We can mimic both results by adjusting the Taq extension temperature. Previous examination of Taq and the Stoffel analog, which lacks 5′ > 3′ exo activity, showed marked differences in temperature dependence of single versus double-stranded template replication 45 that may explain why 5′ > 3′ exo activity matters in some situations but not others. Other polymerase-specific properties like fidelity and processivity were not tested but could also be envisioned to affect blocking. Thus, if certain polymerases are required for a specific application, it is necessary to test them to ensure that they can be effectively blocked by the desired LNAs.

The factors identified here are not guarantees of LNA blocker success, but they provide a guide when designing amplification blockers. The widely different concentrations of the A1AT LNAs required to achieve similar blocking on overlapping sequences highlight the remaining design unpredictability. Blockers of 18–24 nt with alternating LNAs beginning in the second position are a reasonable starting point when selectivity is not critical. If high selectivity for single base changes is needed, shorter oligos should be used (16–18 nt), while longer ones (24 nt) can be used if one or two mismatches are acceptable. For more complete blocking, both DNA strands should be targeted so that neither undesired strand would be synthesized. If only one or a few sequence differences can be exploited and selectivity is needed, it would be best to place mismatches away from the LNA ends.

Materials and methods

Pcr conditions and blocking efficiency.

PCR reactions were carried out with GXL polymerase (Takara), Q5 polymerase (NEB), or GoTaq polymerase (Promega) with the addition of specified LNAs to the listed final concentrations. The target DNA used for Table 1 results was 0.001 ng plasmid DNA 40 . The target DNA used for A1AT in Fig. 5 was Promega human genomic DNA for the wild-type allele or DNA from a human cell line containing the PiZ allele. The target DNA used for all other experiments was synthetic DNA templates (0.001 ng) whose sequences are listed in Table S1 . Most experiments were carried out with the DNA polymerase GXL and cycling parameters of 18 cycles of 98 °C for 10 s, 55 °C for 15 s, and 68 °C for 30 s. In Table 4 , other polymerases were also used with cycling parameters for Q5 of initial denaturation of 98 °C for 30 s followed by 18 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 30 s and a final extension at 72 °C for 2 min. Cycling parameters for GoTaq consist of initial denaturation of 95 °C for 2 min followed by 18 cycles of 95 °C for 30 s, 55 °C for 15 s, and 60 °C or 68 °C for 30 s and a final extension at 60 °C or 68 °C for 5 min. All experiments included controls with no LNA added. Primers, LNAs, and non-degenerate templates were ordered from Integrated DNA Technologies (IDT), and the degenerate template (Template_D) was ordered from Genewiz/Azenta. All template, LNA, and primer sequences are provided in Table S1 . The Template_0 sequence was derived from a plasmid which was used as the template DNA for the reactions in Table 1 . That sequence included a synthetic multicloning region and a region of AAV DNA. PCRs with LNAs, primers, and templates were tested as described in Tables 1 and 2 . For the Table 2 experiments, at least 3 replicates were performed for each LNA/Template combination. From each PCR reaction, 1 µL was run using quantitative capillary electrophoresis according to the recommended protocol for D1000 tapes (Agilent) (Fig. S1 ).

Evaluating primer-LNA binding position

To determine if the distance from the primer to the LNA binding site was important, a forward primer, F3, was designed for use in conjunction with primer R1 and tested with tiling LNAs 1–5 directed to either strand with Template_0. Additionally, two reverse primers, R3a and R3b, were designed and tested in conjunction with primer F1 and tested with LNAs 18_R1, 20_R1, 22_R1, 24_R1, 30_R1, and tiling LNAs 1-4R with Template_0.

Evaluating the base prior to LNA binding region

To investigate whether the base just prior to the LNA binding region was important, two DNAs, Template_A1 (Fig. S3 ) and Template_A2 (Fig. S4 ), were synthesized with a series of single base changes relative to Template_0. With primers F1 and R1 and using conditions stated above, LNAs 18_F1, 30_R1, B4_F1, and B4_R1 were evaluated with Template_A1 and LNAs Tile_1F, Tile_1R, Tile_2F, Tile_3F, Tile_3R, Tile_4F, Tile_4R, Tile_5F, and Tile_5R were evaluated with Template_A2.

Quantitative capillary electrophoresis data analysis

For experiments where Agilent quantitative capillary electrophoresis was used to measure the signal, the concentration was recorded for the band of interest for each sample. Replicates were averaged, and percent blocking was calculated with the formula

Degenerate template sequencing

LNAs 18_F1, 18_F2, 18_R1, 18_R2, 20_F1, 20_F2, 20_R1, and 20_R2, 24D_F2, 24D_R2, and No LNA were evaluated using Template_D where the LNA binding region was synthesized with a degenerate sequence with 79% reference base/7% each non-reference base to determine the role of mismatches in the target sequence. PCR was performed as above, except primers 66Set1 and R1-2 were used. These primers included sequences compatible with Illumina sequencing. Replicates were pooled and purified using Monarch PCR and DNA Cleanup Kit (NEB). A barcoding PCR was performed with 1 µL of each purified PCR1 using GXL polymerase with the following conditions, 98 °C for 2 min, 25 cycles of 98 °C for 10 s, 55 °C for 15 s, 68 °C for 30 s, and a final extension at 68 °C for 3 min. Barcoded products were purified again, and 1 µL run via Agilent Quantitative Capillary Electrophoresis according to the recommended protocol for D1000 tapes to check for clean products and calculate molarity. Products were then pooled equimolarly, and 50% PhiX was added for library complexity. The DNA pool was melted, diluted, and loaded onto an Illumina MiSeq following the standard V2 300 Cycle kit protocol.

Sequencing data analysis

Demultiplexed Fastq files were collected from the instrument, and fastQC was performed to QC the data. Reads were aligned to the reference sequence with Bowtie, and the resulting bam file was filtered to remove secondary and supplementary alignments and unmapped, and poor-quality reads. Next, for each mapped read, each base aligning to the degenerate part of the template was extracted and added to a pivot table, with each row consisting of a readID and the columns containing corresponding individual bases by position. Each base was labeled as R for reference, N for not called, or the base was left unchanged if it did not match the reference. For each readID, the number of mismatches (bases that were not R or N) was calculated in the region targeted by the LNA. The dataset was then separated by the number of mismatches. For each dataset, the number of reference and non-reference bases were counted per position, allowing base frequencies at each position to be calculated.

Data availability

Sequence data is available at SRA with the accession number PRJNA910530. All other data can be found in the manuscript or supplementary files .

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Article Contents

Introduction, materials and methods, acknowledgements.

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Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers

Present address: Joshua D. Levin, KPL, Inc., 910 Clopper Road, Gaithersburg, MD 20878, USA

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Joshua D. Levin, Dean Fiala, Meinrado F. Samala, Jason D. Kahn, Raymond J. Peterson, Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers, Nucleic Acids Research , Volume 34, Issue 20, 1 November 2006, Page e142, https://doi.org/10.1093/nar/gkl756

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Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5′ end (LNA-5′), near the 3′ end (LNA-3′) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, C T ) were characterized using two-way ANOVA. LNA-5′ increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5′ generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3′ and LNA-Even did not improve read lengths or C T . ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence.

Oligonucleotide-based technologies enable better understanding of cellular function and inherited disease. These technologies rely on nucleic acid hybridization for their sensitivity and specificity. However, for a growing number of applications, unmodified oligonucleotides (comprising solely DNA or RNA nucleotides) sometimes yield unacceptable results. Hybridization assays can be difficult to design because important biological features such as SNPs, CpGs, exons, splice sites and protein binding regions are sometimes located in sequence contexts that are poor for design. For example, simple repetitive sequence is poor for primer design, and SNPs may be embedded in short tandem repeats. Features may lie in poor sequence context simply due to the variety of sequence contexts in the genome. Assays may also fail if a method requires hybridization at a defined position relative to the feature of interest. For example, the SNP single base extension method ( 1 ) requires the 3′ terminal base of a primer to be immediately adjacent to the SNP. Methylation Specific PCR similarly requires the bisulfite-treated C of the CpG of interest to be at or near the 3′ end of the primer ( 2 ).

Nucleic acid analogues have a range of hybridization properties that allows modified oligonucleotides to be used successfully in locations where unmodified oligonucleotides fail. Nucleic acid base modifications such as inosine and 7-deazaguanosine reduce melting temperature and have been used to improve primers in GC-rich templates ( 3 ). One of the most promising analogues used to raise melting temperature is Locked Nucleic Acid (LNA). LNA is a bicyclic ribose derivative with a bridging methylene group between O-2′ and C-4′. LNA provides the largest known increase in thermal stability of any modified DNA duplex ( 4 ), because it reduces the unfavorable entropy of duplex formation and may improve base stacking. LNA also has greater mismatch sensitivity than DNA ( 5 , 6 ). LNA is available for all four bases, so it is easily placed at any position in an oligonucleotide.

LNA has been used in many applications. SNP applications include: TaqMan ® ( 7 ); Molecular Beacons ( 6 ); fluorescence polarization ( 5 ); and immobilization ( 8 ) probes. A partial list of other applications includes PCR primers ( 9 ), allele-specific PCR ( 10 ), real-time PCR probes ( 11 , 12 ), antisense oligonucleotides ( 13 ), siLNA duplexes ( 14 , 15 ), single molecule miRNA detection ( 16 ), miRNA probes ( 17 ), decoy oligonucleotides ( 18 ), microarray probes ( 19 ), aptamers ( 20 ) and sequence detection using piezo-resistive cantilevers ( 21 ).

Effective design of LNA oligonucleotides requires accurate thermodynamic parameters ( 14 ). Our group recently determined sequence-dependent parameters for single, internal LNA incorporation into DNA duplexes ( 22 ). The effects of LNA on duplex stability are highly sequence-dependent. Moreover, the stabilization may be either entropic or enthalpic, depending on sequence context ( 22 ). These parameters provide precise estimates of duplex melting temperature. Their incorporation into Celadon's ProbeITy™ oligonucleotide assay design software enables the design of LNA primers and probes.

Effective design also requires positional rules ( 14 ). Latorra et al. ( 9 ) provided the first systematic study of the effect of LNA position and PCR conditions on LNA primer efficiency. They modified a single primer sequence with 11 LNA patterns and used the intensity of final product bands as the measure of priming efficiency. They found that LNA should be spaced evenly across the primer and that no more than three LNA should be used. This research design does, however, confound LNA number with its pattern of incorporation and real-time PCR would provide a more accurate and precise estimate of any LNA effects. Exiqon, the intellectual-property holder of LNA, does not make positional recommendations at this time on their web site.

The goal of the present study is to generate LNA positional design rules for sequencing and PCR primers. The effects of LNA on the two methods are expected to be similar. Our research design was to first identify a number of short DNA primers that generated poor sequencing reads. We then modified each primer with three different patterns of LNA incorporation (LNA-5′, LNA-Even and LNA-3′) with the goal of testing which patterns improve performance. Performance is assessed quantitatively using sequencing base call quality scores and PCR cycle thresholds. ANOVA enabled us to assess the magnitude of the effects of different LNA incorporation patterns and it enabled for the first time the statistical testing of interaction between primer sequence and LNA pattern. Significant interaction would mean that a particular pattern of LNA incorporation improved the performance of some primers, but degraded the performance of others.

We found that the LNA-5′ incorporation pattern significantly improved both sequencing and PCR primers, but LNA-3′ and LNA-Even did not. These results probably arise because LNA near the 5′ end enhances stability without increasing mispriming, while LNA near the 3′ end stabilizes mispriming, although our results cannot exclude the possibility of direct LNA effects on polymerase extension. The ANOVA research design also provided objective evidence for interaction, meaning that the effect of LNA incorporation depends on primer sequence.

Template and primer design and synthesis

The DNA sequencing templates were PCR amplicons from the human membrane cofactor protein (MCP) gene (AL035209) and the human ataxia telangiectasia mutated (ATM) gene (AP001925). All PCR and sequencing primers were designed using ProbeITy™ oligonucleotide assay design software (Celadon Laboratories, Inc., Hyattsville, MD; www.celadonlabs.com ). Unmodified DNA sequencing primers were shortened progressively to derive low- T M , poor-quality sequencing primers, as described in Results. Placement of LNA into the primers was guided by prototype software that implemented a database of thermodynamic parameters for single LNA incorporations ( 22 ). DNA and LNA-enhanced primers (all from IDT, Coralville, IA) were suspended in 1× TE at ∼100 μM and concentration was measured using absorbance.

PCR to generate sequencing templates was performed in 10–50 μl reactions using 200 nM of each PCR primer, 165 ng genomic DNA (Coriell, Camden, NJ) and 1.25 U AccuPrime Taq DNA polymerase with AccuPrime buffer II, which includes dNTPs (Invitrogen, Carlsbad, CA). Thermal cycling began with denaturation at 94.0°C for 2 min followed by 30 cycles of 94.0°C for 30s, 60.0°C for 30s and 68.0°C for 80s. PCR product was visualized on 1.5% agarose gels with ethidium bromide. PCR products were purified using Centricon-100 micro-concentrators (Millipore, Bedford, MA).

DNA sequencing reactions

Sequencing reactions on purified PCR product templates were performed by the DNA Sequencing Facility at the Center for Biosystems Research (CBR), University of Maryland Biotechnology Institute. The ABI Big Dye Terminator DNA Sequencing kit (V. 3.1) and ABI thermal cyclers were used, with an annealing temperature of 50°C. Sequencing traces were produced on the ABI 3730 96-channel, 50 cm array DNA Analyzer and base quality scores were generated using the Phred algorithm included with the ABI 3730 software ( 23 , 24 ). To ensure that all samples were properly tracked, every trace was aligned with its intended target sequence using Sequencher (GeneCodes, Inc., Ann Arbor, MI). After an array upgrade early in the project, replicates performed in the same sequencing batch and control samples that were run on different days exhibited negligible variation.

Quantitative assessment of sequence read quality

For hypothesis testing for the ANOVA research design, Phred counts were selected as a quantitative measure of sequencing read quality. Phred is an algorithm ( 23 , 24 ) that generates sequence reads by calling bases in the trace and assigning an estimated error probability P and a quality score Q to each call. The values are related by Q = −10[log 10 ( P )]. A Phred Q score of 20 equates to a 99.0% probability that the base-call is correct and a Phred Q score of 30 is a 99.9% probability. Phred Q = 20 is the threshold quality standard for genome sequencing projects [Ref. ( 25 ) and http://www.genome.gov/10000923 ].

Our quantitative measures of sequencing quality are the numbers of bases that were assigned Phred Q scores ≥20 or ≥30 in the first 1000 bases of DNA sequence. We define these numbers of bases as the Phred Q20 and Q30 counts. For sequencing where the quality remains high until near the end of the usable data, the Phred Q30 score is essentially the read length. In our data, Phred Q20 and Q30 counts were highly correlated ( r 2 = 0.985), so we generally report only the Q30 counts. We observed a good qualitative correlation between high Phred Q20 and Q30 counts and subjectively robust sequencing reads, those with strong fluorescent peaks and little or no overlap. Noisy electropherograms with overlapping fluorescent peaks generated low Phred Q20 and Q30 counts. (See the sample electropherograms in Figure 1 .)

Representative effects of LNA incorporation pattern on sequencing electropherograms. Portions of electropherograms generated from ATM2 primers with the indicated LNA substitution patterns are shown. The same AT-rich ATM PCR product was the template for all four reactions and all reactions were performed in the same plate. The primers used are as follows: (A) unmodified ATM2; (B) ATM2-LNA-Even; (C) ATM2-LNA-5′; (D), ATM2-LNA-3′. The panels are aligned at A230 as indicated by the dashed line.

Representative effects of LNA incorporation pattern on sequencing electropherograms. Portions of electropherograms generated from ATM2 primers with the indicated LNA substitution patterns are shown. The same AT-rich ATM PCR product was the template for all four reactions and all reactions were performed in the same plate. The primers used are as follows: ( A ) unmodified ATM2; ( B ) ATM2-LNA-Even; ( C ) ATM2-LNA-5′; ( D ), ATM2-LNA-3′. The panels are aligned at A230 as indicated by the dashed line.

Real-time PCR with SYBR green detection

Real-time PCR was performed in 96-well plates with optical caps using a GeneAmp 5700 (Applied Biosystems). Reaction volume was 50 μl, including a SYBR Green PCR Master Mix that included enzyme, Mg 2+ and dNTPs (ABI, Foster City, CA), 100 nM each PCR primer and 0.5 ng/μl genomic DNA. A hot start of 2 min at 50°C and 10 min at 95.0°C was followed by 40 cycles of 95.0°C for 15 s and 60.0°C for 60 s. The identities of the PCR products were confirmed using restriction analysis. For comparison of different forward primers, master mixes were used that included all the other components, including the appropriate reverse primer. The cycle threshold ( C T ), defined as the thermal cycle at which the signal intensity surpassed a value of 0.1, was used as the quantitative measure of real-time PCR quality.

A dissociation curve from 60.0 to 92.0°C was performed following each PCR run. All primer sets used in this study exhibited a single peak between 70.0 and 75.0°C. This relatively low amplicon melting temperature is due to their AT-rich composition. Control reaction chambers that contained the master mix with only the reverse primer did not generate a peak in the dissociation curve, indicating that the reverse primer alone did not generate genomic product or signal due to primer-dimers. Control standard PCR showed no product in the absence of template.

Prediction and measurement of primer melting temperatures

To predict the melting temperature of the DNA primers under PCR conditions we assumed 50 mM KCl, 1.8 mM MgCl 2 , 1 mM dNTPs and 200 nM total primer concentration. The unified ΔH° and ΔS° nearest-neighbor parameters of SantaLucia ( 26 ) and a standard salt correction formula ( 27 ) were used. To predict melting temperatures under thermal melt curve conditions we assumed 1 M NaCl and 2 μM total primer concentration, typically giving T M values 10–15°C higher. UV absorbance versus temperature curves were obtained and analyzed as previously described ( 22 ).

To predict melting temperature for the DNA–LNA primers we added the LNA sequence-dependent ΔΔH° and ΔΔS° parameters from Ref. 22 to the ΔH° and ΔS° values obtained as above. For 5′-terminal LNA substitutions, we treated the terminal LNA the same as an internal modification. This analysis ignores possible differences between internal and terminal positions. We also ignore possible cooperative effects between multiple LNAs within a sequence. This assumption that the thermodynamic effects of each LNA are additive and independent is unlikely to be correct. The effect of additional LNA on T M plateaus, so subsequent incorporations have smaller effects than the initial one ( 28 ).

The research design was a two-factor, mixed-model ANOVA with replication. The two factors were LNA position and primer sequence. The mixed-model aspect derives from the fact that chemistry (LNA position) is a treatment effect fixed by us, while there was no such experimental manipulation of primer sequences after their initial selection.

Calculations were performed using the ‘ANOVA-Two-Factor with Replication’ Analysis ToolPak add-in of Microsoft Excel. Certain F -tests were performed manually, because Excel computes F -statistics by dividing all mean squares by the error mean square regardless of the statistical significance of the interaction. However, when the interaction is significant, as it was in our data, the correct F -statistic computation for a mixed model is to divide the fixed treatment effect mean square (in our case, LNA pattern) by the interaction mean square (see Ref. 29 , pp. 337–338). ANOVA assumes that data are normally distributed. Our data provide insufficient power to reject the normal distribution regardless of its true distribution. However, variation in the Q30 count is likely to have a normal distribution.

The first ANOVA procedure analyzed differences in the mean Q30 count among all LNA patterns. This analysis was globally informative of differences among means, but it was not informative of which mean or groups of means were statistically different from other means or groups of means. To analyze these differences required comparisons, which are indicated only when the global analysis is significant. Possible comparisons among LNA patterns include six pair-wise comparisons and there were also comparisons among groups of means, such as the unmodified primer versus the three LNA patterns. The decision on which comparisons to perform was influenced by the available degrees of freedom, which in this case is three because there are four chemistry patterns.

Since our basic premise was that the LNA patterns would generate significantly different Q30 scores when compared to the unmodified primers, if the global analysis was found to be significant the obvious choice was to make pair-wise comparisons of mean Q30 score between each of the three LNA patterns and the unmodified primer. These three planned comparisons consume, but do not exceed, the available three degrees of freedom. P -value corrections for multiple comparisons were not performed because these are only necessary when the number of comparisons exceeds the degrees of freedom.

Experimental design

LNA incorporation is most likely to be necessary for improving the performance of short, low- T M , AT-rich primers. Using six such primers, we sought to identify positions in sequencing primers at which LNA has a statistically significant effect on sequencing quality, either positive or negative. Our expectation was that at least one LNA pattern would lead to higher sequencing quality, because LNA increases primer melting temperature. Three LNAs were incorporated into each primer, because this was sufficient to elevate the predicted melting temperatures into the range that produced good quality sequencing reads. Previous worked showed that PCR failed when primers had more than three LNAs ( 7 ).

LNA was distributed across the test primers in three different patterns. The primer sequences were divided into three segments of equal length: 5′, middle and 3′. The ‘LNA-5′’ pattern had two LNAs in the 5′ section and one in the middle. The ‘LNA-Even’ pattern had one LNA in each section. The ‘LNA-3′’ pattern had one LNA in the middle and two in the 3′ section. This research design, using four primers with identical base sequences but differing in composition, isolates the effect of LNA. Sequencing reactions for each primer were performed in triplicate to generate within-primer variances. This approach allowed us to assess the amount of variation that was due to interaction between primer sequence and LNA pattern. We applied the same research design to PCR primers.

Selection and characterization of poor unmodified DNA sequencing primers

Primers were designed for two arbitrarily chosen realistic test cases, the AT-rich human disease genes (MCP/CD46) and ATM. MCP is a receptor for a wide variety of pathogens ( 29 ) and ATM is a central regulatory gene in the cellular response to DNA damage ( 30 ). We expect that the results obtained on these primers will generalize to all primers that have a melting temperature that is low with respect to the sequencing reaction temperature.

To obtain low- T M primers that generated poor sequencing quality, for each of the two genes three sets of AT-rich primers that had predicted T M s of ∼60°C were initially identified. One base at a time was removed from each primer's 5′ end until a primer with a predicted T M of ∼35°C was reached, which should be sufficiently low to ensure poor sequencing. From each of the six resulting sets of primers, about six primers were selected that sampled melting temperature every 3–4°C (sequences not shown). Triplicate sequencing reactions were performed for each selected primer. From each of the six sets, the highest- T M primer that produced a poor sequencing read was chosen, resulting in six primers of Table 1 , which ranged in predicted melting temperature (under sequencing conditions) from 34.0 to 40.0°C. These primers (ATM1, ATM2, ATM3, MCP1, MCP2 and MCP3) were modified with the LNA-5′, LNA-3′ and LNA-Even patterns.

DNA sequencing primers used to test LNA incorporation patterns

The six low- T M primers derive from the MCP and ATM genes.

Phred Q20 and Phred Q30 counts are defined as the total number of bases out of the first 1000 bases in the sequencing electropherograms that have Phred scores of atleast 20 or 30 respectively. The average and standard deviation of three reactions is given.

The T M (in °C) is predicted for sequencing reaction conditions: 50 mM KCl, 50 mM NaCl, 1.8 mg Mg 2+ , 100 nM total (primer), 1 mM total dNTPs.

This row refers to earlier sequencing, during the sequence selection/design phase of the work, using the MCP1 primer under less optimal conditions.

After the LNA-modified primers were chosen and synthesized, the sequencing center upgraded the capillary array and sequencing procedures, which markedly improved the sequencing with the chosen MCP primers. We report results from the MCP1 primers from both before and after the upgrade in Tables 1 and 3. All the other results and all of the statistical analyses below for the ATM and MCP primers are for the upgraded array. The long and consistent post-upgrade MCP reads of Table 1 contrast with the short and variable ATM reads. This provided us with the opportunity to test the effects of LNA on primers of various read lengths and consistency.

T M predictions for LNA-enhanced primers

To test melting temperature predictions for LNA-modified primers, we measured the T M of each sequencing primer using standard melt conditions ( 22 ); see Table 2 . Predictions for DNA primers were highly accurate: they deviated <±1.0°C for five of the six primers. The T M for ATM3 (CCAGAAAGCCA) was 2.6°C higher than predicted. This could be due to the short A-tract, which is known to generate aberrant melting behavior ( 31 ). For the LNA-substituted primers, the average difference between predicted and observed was also small, just +0.8°C. However, this average result masks a decrease in accuracy as compared to the all-DNA primers: the difference between observed and predicted ranged from −3.7° to +5.6° and 11 of the 17 primers had differences >±1.0°. This confirms that the single-LNA substitution results from ( 22 ) must be extended in order to describe multiple incorporations.

LNA-substituted sequencing primers

LNA positions are underlined and preceded by + signs.

The T M is predicted for the sequencing reaction conditions specified in the legend for Table 1

The Δ T M is the predicted minus observed under melt conditions.

The LNA-5′ incorporation pattern dramatically improves DNA sequencing

LNA incorporation can improve primer performance substantially, and the effect is markedly dependent on its pattern of incorporation ( Table 3 and Figures 1 and 2 ). The electropherograms of Figure 1 show that the primary cause of low Phred Q30 and Q20 counts for the poorly performing ATM primers was multiple sequence ladders. Multiple ladders arise from mispriming, so the observed effects cannot be due to inhibition of polymerase extension by LNA; although LNA may well have some direct effect on the polymerase, primers with LNA at the 3′ position have been used for SNP detection by PCR ( 32 ). LNA-5′ nearly eliminated the mispriming that made the sequence unreadable for the other primers. Table 3 shows that the improvement is reflected in an average Phred Q30 count 60% higher than the average unmodified primer, as well as a decreased variance in the Q30 count. The effect of LNA-5′ was more pronounced for the ATM primers (for which the Q30 count nearly quadrupled) than for the MCP primers, because the unmodified MCP primers already had high Q30 counts, but in no case did LNA-5′ decrease performance. The results ( Tables 1 and 3 ) from earlier sequencing reactions on the MCP1 primer confirm the superiority of the LNA-5′ pattern. In contrast, LNA-Even and LNA-3′ did not improve performance, yielding substantial mispriming and average Phred Q30 counts that were slightly less than those of unmodified primers. The same order of quality—LNA 5′ > Unmodified DNA > LNA 3′ > LNA-Even—was also seen in the Phred Q20 counts (data not shown). The LNA-5′ effect was presumably due to an increase in hybridization strength at the 5′ end of the primers that stabilized on-target hybridization and destabilized off-target 3′ mispriming.

The effects of LNA incorporation pattern on sequencing read quality. The numbers of bases whose Phred scores exceed 30 (the Phred Q30 counts) within the first 1000 bases read are shown. Each of the four LNA incorporation patterns was applied to each of the six AT-rich sequencing primers as indicated below the graph; the data are from Table 3. The order of presentation within each primer group, from left to right, is Unmodified DNA (light gray); LNA-3′ (dark gray); LNA-5′ (white); LNA-Even (black).

The effects of LNA incorporation pattern on sequencing read quality. The numbers of bases whose Phred scores exceed 30 (the Phred Q30 counts) within the first 1000 bases read are shown. Each of the four LNA incorporation patterns was applied to each of the six AT-rich sequencing primers as indicated below the graph; the data are from Table 3 . The order of presentation within each primer group, from left to right, is Unmodified DNA (light gray); LNA-3′ (dark gray); LNA-5′ (white); LNA-Even (black).

Phred Q30 counts for sequencing with unmodified and LNA-substituted primers

The average and standard deviation of the Phred Q30 Counts are given from three sequencing runs. See Table 2 for primer sequences.

The data in this row are from earlier sequencing reactions done under non-optimal conditions. Two sequencing runs were performed for each LNA-modified primer. These results are not included in the averages below. n.d., not done.

ANOVA shows that the LNA-5′ effect is statistically significant

The ANOVA results ( Table 4A ) show that primer sequence [ F (5,48) = 166.9, P < 0.0001, where F (5,48) is the F -test statistic for 5 d.f. and 48 observations], LNA pattern [ F (3,15) = 8.2, P = 0.0018] and interaction between primer sequence and LNA pattern [ F (15,48) = 14.0, P < 0.0001] all have a highly significant effect on the Phred Q30 count. A significant effect of primer sequence is not unexpected. Primers of similar base composition but different base order often generate disparate Q30 counts or PCR product. This can be due to secondary structure, off-target hybridizations and the ability of the primer to be an efficient substrate for the polymerase. The significant interaction effect means that the effect of LNA incorporation on Q30 count depends on the primer sequence to which the pattern is applied.

ANOVA of LNA effects on DNA sequencing and PCR

(A) Primer sequence, LNA chemistry and an interaction between the two all significantly affect the Q30 count, as indicated by the P -value column. The underlying data is that of Table 3 .

(B) The underlying data is that of Table 5 .

SS = Sum of squared deviations from the mean.

d.f. = Degrees of freedom.

MS = Mean square, SS/d.f.

F = F -test statistic.

F crit = the F -distribution critical value for achieving significance at P = 0.05.

Since the global test also identified a significant effect of LNA pattern on Q30 count, pair-wise comparisons were performed in order to determine which LNA patterns were significantly different from the unmodified DNA. The paired comparisons confirmed that the LNA-5′ primers generated significantly longer Q30 counts than the unmodified primers [ F (1,5) = 6.58, P = 0.050; data not shown]. However, the differences between the LNA-3′ primers and the unmodified primers [ F (1,5) = 1.47, P = 0.280] and the LNA-Even primers and the unmodified primers [ F (1,5) = 3.76, P = 0.110] were not significant (data not shown). Statistically, this lack of significance could be due to the small sample size and the large amount of interaction.

Selection and characterization of poorly-performing unmodified PCR primers

To generate test DNA primers that yielded poor PCR results, we started with the six AT-rich primers described above and extended them to the 5′ side to give primers with predicted melting temperatures of ∼60.0°C, denoted with an ‘ L ’ at the end of the primer name. Due to the AT-rich nature of the target sequence, we were able to identify reverse primers for only three of the forward primers, MCP1L, ATM1L and ATM2L. We then performed real-time PCR with SYBR Green detection using sets of primers differing by 1 bp, as for DNA sequencing. These experiments identified the threshold forward primer that provided good PCR efficiency [denoted DNA( n + 1) in Table 5 ] and the next-shorter forward primer, DNA( n ), which provided poor PCR efficiency.

Real-time PCR is enhanced by the LNA-5′ incorporation pattern

The primers are extended versions of those in Table 1 .

PCR conditions are as described in Materials and Methods.

The tabulated values are the cycle threshold ( C T ) averages and standard deviations from three replicate real-time PCR experiments ( Figure 3 ). C T values in parentheses indicate values that are unreliable and were not used in quantitative analysis.

We then incorporated LNA into the three unmodified DNA( n ) primers using the LNA-5′, LNA-3′ and LNA-Even patterns as described above. The goal was to elevate each melting temperature to that of the DNA( n + 1) primer. This required three LNAs for the ATM2 LNA-3′ primer and two LNAs for all the others. We then performed real-time PCR experiments in triplicate with each of the five primers. All replicates were performed in the same plate. PCR experiments with just the master mix, containing the reverse primer, generated a baseline signal. As before, statistical analysis was by two-way ANOVA with replication.

The LNA-5′ incorporation pattern improves real-time PCR

Representative real-time PCR results are shown in Figure 3 and Table 5 . The amplification plots of Figure 3 show SYBR Green fluorescence as a function of PCR cycle. Table 5 shows the corresponding cycle threshold ( C T ); considering PCR amplification efficiency instead gave qualitatively consistent conclusions, but the C T results are discussed here because they captured the qualitative observation that many of the amplification reactions never reached signal levels comparable to the on-target amplification. Figure 3A shows that for the MCP1L primers, the LNA-5′ and LNA-Even primers worked about as well as the efficient DNA( n + 1) primer. All three primers plateau at about the same level and cycle. In contrast, the LNA-3′ primer and the poorly-performing DNA( n ) primer exhibited much less amplification. The C T values for these primers were actually higher than in the master mix control, which contained only the reverse primer. C T values of this magnitude are likely to be spurious and very low levels of amplification were observed.

Real-time PCR using LNA-modified versus DNA primers. SYBR green fluorescence intensity (arbitrary units) is shown as a function of cycle number. (A) Shows the MCP1L forward primers and (B) shows ATM1L. The templates are PCR amplicons of the corresponding genes. The data points are the averages and standard deviations of triplicate reactions. For presentation on a log scale, values <0.01 (all were near background, ≥−0.06) are displayed at 0.01, but with their actual standard deviations. The forward primers are as follows: open circles, DNA (n + 1); squares, DNA(n); filled circles, LNA-3′; upward-pointing triangles, LNA-5′; diamonds, LNA-Even; downward-pointing triangles, no forward primer.

Real-time PCR using LNA-modified versus DNA primers. SYBR green fluorescence intensity (arbitrary units) is shown as a function of cycle number. ( A ) Shows the MCP1L forward primers and ( B ) shows ATM1L. The templates are PCR amplicons of the corresponding genes. The data points are the averages and standard deviations of triplicate reactions. For presentation on a log scale, values <0.01 (all were near background, ≥−0.06) are displayed at 0.01, but with their actual standard deviations. The forward primers are as follows: open circles, DNA ( n + 1); squares, DNA( n ); filled circles, LNA-3′; upward-pointing triangles, LNA-5′; diamonds, LNA-Even; downward-pointing triangles, no forward primer.

Figure 3B shows that the DNA( n + 1) ATM1L primer outperformed all of its related LNA primers. The signal strengths of all but the ATM1L DNA( n + 1) primer are relatively low, as evidenced by C T values of >30, compared to C T values of 20–25 for a typical efficient PCR. The low signal strengths could be due to low primer melting temperatures of the forward primers (∼46.0–52.5°C) compared to the reaction temperature (60.0°C). Also, the melting temperature of the forward primers is much lower than the melting temperature of the reverse primers (∼60.0°C). This disparity may contribute to inefficiency. The 5′-LNA primer was, however, clearly superior to LNA-3′, LNA-Even and the DNA( n ) primer. These latter reactions had high C T values, comparable to the MCP1L LNA-3′, LNA-Even and master mix. The ATM2L primers displayed a pattern similar to that of the ATM1L primers ( Table 5 ).

The general picture that emerges from these data is that incorporating LNA at or near the 5′ end of a PCR primer improves short PCR primers. The LNA-5′ pattern improved all of the DNA( n ) primers, although not always to the same level as the DNA( n + 1) primers. It might be possible to improve the performance of the ATM1L and ATM2L LNA-5′ primers to the level of the DNA( n + 1) primer by adding additional LNAs and/or by using a different pattern of LNA-5′ incorporation. The real-time PCR results on the ATM primers demonstrate that raising primer T M is not sufficient to ensure improved priming performance. We suspect that the ATM2L LNA-5′ primer may exhibit self-hybridization that hampers priming: given that LNA-G: DNA-T can form a stable mismatch ( 33 ), the + G CA+ T ATAAGT portion could form a 10 bp helix with two internal A–A mismatches. It is also possible that PCR amplification is inhibited through LNA's effects on the polymerase, but as for sequencing, it appears that the LNA primers are capable of being extended even when they do not give amplifiable product.

The effect of the LNA-5′ pattern on PCR primers is significant

ANOVA was applied to the factors affecting cycle threshold ( Table 4B ). Since the high- C T primers generated little or no specific product, the C T values for these primers are unreliable. For primers where at least one replicate failed to attain the intensity threshold after 40 cycles, we do not report the standard deviation in Table 5 . For ANOVA, we replaced these C T values with a value of 40.

As observed for the sequencing primers, PCR primer sequence [ F (2,30) = 45.38, P < 0.0001], LNA pattern [ F (4,8) = 4.31, P = 0.038] and interaction between primer sequence and LNA pattern [ F (8,30) = 11.16, P < 0.0001] each have a significant effect on the PCR amplification. A significant effect of primer sequence on PCR is not unexpected, for the same reasons discussed above. The amplification plots showed that it was necessary to perform pairwise comparisons only for the LNA-5′ pattern, which revealed that the LNA-5′ primers generated significantly lower C T values than DNA( n ) [ F (1,2) = 21.35, P = 0.044].

Interaction between primer sequence and LNA pattern was highly significant in the global analysis and almost significant [ F (2,12) = 3.00, P = 0.088] in the pair-wise comparison between LNA-5′ and the unmodified primer described above. Although the interaction was not quite significant in the pair-wise comparison, we conservatively computed the LNA-5′ versus unmodified pair-wise F -statistic by dividing the LNA pattern mean square by the interaction mean square, as recommended by Sokal and Rohlf ( 34 ) and described in Material and Methods.

Effect of LNA position

The sequencing and quantitative PCR results show that LNA-5′ is the best incorporation pattern for both methods. LNA-5′ primers were never worse than their unmodified counterparts. They nearly quadrupled the sequencing read lengths, as measured by Phred Q30 counts, of three primers designed for the ATM gene and they also generated more consistent Q30 counts. The LNA-5′ pattern also increased PCR yield relative to unmodified DNA of the same length. In contrast, the LNA-3′ and LNA-even incorporation patterns generated lower Q30 counts and higher C T values than the unmodified primers, due in part to multiple sequencing ladders and non-specific PCR products. The LNA-Even appears to be worse than the LNA-3′ pattern, but the differences were not statistically significant.

These findings are sensible in view of the thermodynamics of primer hybridization and the mechanism of sequencing and PCR. In general, a primer that binds more strongly at its 5′ end should have better specificity than a primer that binds more strongly at its 3′ end. Partial off-target hybridization at the primer's 5′ end is not expected to generate product, but partial hybridization at the 3′ end may result in polymerase extension and thus non-specific product. Experimentally, Rychlik ( 35 ) found that high efficiency primers often have both 5′ hybridization that is stronger than 3′ hybridization and also moderate-strength 3′ hybridization. The 5′ partial hybridizations out-compete the 3′ partial hybridizations, while the moderate 3′ hybridization keeps the 3′ end of the primer hybridized to its target. When the GC% of the 3′ end becomes too high, the primers are susceptible to off-target priming.

Since LNA-5′ primers presumably hybridize more strongly at their 5′ ends, on-target hybridization is stabilized but off-target hybridization does not lead to product. In contrast, the LNA-3′ and LNA-Even primers stabilize off-target hybridization leading to non-specific product. They may also reduce on-target yield by diverting polymerase away from the target site. It is unlikely that the poor performance of the LNA-3′ and LNA-Even primers was due to inhibition of polymerase extension ( 36 ). In this case, clean but low-amplitude sequencing traces would be expected (low signal). Instead, we observed strong and jumbled traces (high noise), indicating false priming rather than inhibition.

Our finding that LNA-Even is the worst pattern is in contrast with Latorra's ( 9 ) conclusion that LNA-Even is best. We did observe that LNA-Even primers performed well some of the time and this may have been the case for the single primer that they studied. Finally, our finding that LNA-3′ primers performed less-well than conventional primers is consistent with Latorra's results ( 9 ).

The evidence suggests that LNA should never be placed near the 3′ end of sequencing primers or PCR primers unless it is necessary for methods like allele-specific PCR, for which LNA at the variable position has been reported to enhance allelic discrimination ( 10 ). This improved discrimination derives in part from LNA's destabilization of mismatches and in part from a decreased efficiency of the polymerase at an LNA end, as evidenced by an overall weaker signal as compared to perfect-match primers.

ANOVA research design

The decision to use ANOVA in the early stages of experimental design forced us to plan data analysis in advance, whereas otherwise it would not have been obvious how to fully populate the research design. The use of ANOVA here helped make it clear that the proper comparison of LNA primers is to unmodified primers of the same sequence, which was not done in several previous studies ( 9 , 14 , 15 , 37 ).

ANOVA has much to recommend it over the more commonly used paired t -test. While the two methods are mathematically equivalent for pair-wise comparisons, the paired t -test cannot provide a global analysis when there are more than two experimental conditions. The recommended method for ANOVA is to perform pair-wise comparisons only after the global analysis indicates significance. The paired t -test is not informative as to interactions among experimental dimensions and it does not provide the structure to objectively determine when to adjust P -values for multiple comparisons. ANOVA also provides guidance on the needed sample size and power given initial expectations for the magnitude of an effect.

In this study, we used Phred quality scores and cycle threshold ( C T ) as quantitative measures of primer quality ( 38 ). These measures may be more accurate and precise than previous measures, such as read length or gel band density, and they are likely to become more widely-used in developing primer design rules. The use of triplicate data on a number of primer sequences is an expensive research design, but the resulting decrease in the variance added to the statistical power of the ANOVA. This depth of data coverage was made possible by defining a limited number of LNA patterns. The ANOVA allowed us to formally test for interactions between primer sequences and LNA incorporation pattern and distinguish it from the main effect of LNA incorporation pattern. The interaction was statistically significant and was not qualitatively obvious, and this work solidifies previous anecdotal evidence for such interactions ( 9 , 14 , 15 , 37 ).

Additional thermodynamic considerations for the design of LNA primers

The ideal thermodynamic dataset for use in primer design algorithms would be a complete description of all possible LNA–DNA mixmers hybridized to their complements and to all possible mismatches. Such a dataset is not available, but existing data allows us to propose some general rules for LNA-enhanced primer design.

Our database of thermodynamic parameters for single, internal LNA incorporation ( 22 ) generated melting temperature predictions that were accurate enough to be useful for primer design, but not as accurate as the predictions for unmodified primers. In general, LNA pyrimidines provide more stabilization than purines, with LNA-A being the least stabilizing base. The most inaccurate predictions tended to occur in primers that have LNA in terminal or penultimate positions. These results suggest that the generation of thermodynamic parameters for LNA incorporation at terminal and penultimate positions will enable accurate T M predictions for a larger variety of primers. Preliminary results (M. F. Samala, J. Levin, R. J. Peterson and J. D. Kahn, unpublished data) suggest that 5′ terminal LNA provides only a slight increase in stability and the 5′ penultimate position less stabilization than internal positions. The accuracy of T M predictions should also increase with the availability of thermodynamic data for multiple LNA incorporations and for mismatches. While mismatch data are obviously relevant to SNP methods, they are also useful for predicting LNA stabilization of off-target hybridization. In this regard, LNA-G:DNA-T mismatches appear to be especially problematic ( 33 ).

Accurate prediction of melting temperature for primers that contain any modified chemistry at any position will require generation of a comprehensive database of thermodynamic parameters: single and multiple internal incorporations; 5′ and 3′ terminal and penultimate incorporations; and mismatches. Thermodynamic data will improve design for any method that uses hybridization of modified nucleic acid chemistries.

Applications of LNA primers

The design of LNA primers can be complicated because of the stabilization of both on- and off-target hybridization. Optimal LNA primer design will require assay design software that applies positional and thermodynamic rules. Moreover, it is important to establish the conditions for which LNA is beneficial, since it is substantially more expensive than traditional DNA and RNA chemistries. Here we consider only the thermodynamic aspects of LNA use; of course, its ease of synthesis, chemical stability and resistance to nucleases are also critical in many applications.

LNA is likely to improve performance most significantly when primer hybridization would otherwise be inefficient. This occurs most frequently in AT-rich regions, where unmodified primers of limited lengths have melting temperatures that are too low to hybridize well under standard reaction temperatures. LNA incorporation should then improve sequencing quality and PCR yield and we have demonstrated that poorly-performing primers can be rescued most effectively using the LNA-5′ incorporation pattern. LNA might also be useful for sequencing GC-rich templates on which the available priming site is short, as it might be able to increase primer T M to allow the use of higher primer annealing and extension temperatures without increasing primer length.

LNA-modified short primers can be positioned with more versatility than DNA primers. Exact placement is essential to methods that target a specific feature with stringent primer-placement rules, such as MassArray SNP genotyping ( 1 ) and pyrosequencing of potentially methylated CpG sites ( 39 ). Short primers are better able to avoid tandem and interspersed repeats, as well as sequences that generate primer hairpins or primer-dimers. These regions have been problematic for the new sequencing technologies.

LNA incorporation near the 5′ end of sequencing and PCR primers improves performance, but LNA near the 3′ end and LNA evenly spaced throughout the primers do not. We suggest that LNA-5′ increases primer stability at its target site without enhancing extension of primers at off-target sites, whereas LNA-3′ and LNA-even promote mispriming. ANOVA methodology enabled us to rigorously quantify the effects of primer sequence, LNA position and the interaction between the two. The positional rules derived here, as well as thermodynamic parameters for any kind of LNA incorporation, are essential for software that designs LNA primers for sequencing and PCR.

We are grateful to Suwei Zhao, Kongyi Jiang and Tigist Edeto of the CBR DNA Sequencing Facility for efficient and high-quality sequencing. Dr Louisa Wu of CBR provided access to the GeneAmp 5700 real-time PCR instrument. Dr David Schuster (Quanta Biosciences, Gaithersburg, MD) and Subhamoy Pal (CBR) provided assistance with real-time PCR. We are grateful to Larry Kessner of Celadon Laboratories for his ability to see the big picture. This research was funded by a NCI SBIR contract (PHS 2004-1, Topic 191, #N43-CB-56000). Funding to pay the Open Access publication charges for this article was provided by the SBIR contract.

Conflict of interest statement. Celadon Laboratories sells primer design software and services that consider LNA-modified oligonucleotides.

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Design and performance analysis of low power LNA with variable gain current reuse technique

  • Published: 08 May 2021
  • Volume 108 , pages 351–361, ( 2021 )

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  • Dheeraj Kalra 1 , 2 ,
  • Vishal Goyal 2 &
  • Mayank Srivastava 1  

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This paper presents a CMOS low power Variable Gain Low Noise Amplifier for 26–34 GHz in 45 nm process technology, which composes of cascaded complimentary common gate (CCG) stage and digital current steering amplifier. First stage is CCG stage, which helps in achieving the low power consumption and less area. Second stage is variable gain amplifier, uses current reuse technique as well as g m -boost technique and has constant dc current to make the input impedance stable. Source degeneration technique cancel out MOS parasitic capacitance help in achieving linearity. Simulated maximum peak gain is 13.139 dB at 30.57 GHz and lowest peak gain is 7.75 dB at 26 GHz i.e. approximately flat over the entire band. Lowest NF is 3.08 dB at 32.6 GHz. Process corner simulation has been done for all four corners (S–S, S–F, F–S, F–F) showing robustness of LNA. Input return loss has value less than − 9.58 dB while output return loss has less than − 2.6 dB showing good matching; power consumption is 16 mW for dc current of 16 mA at 1 V. MOS active chip area is 76.727 µm 2 .

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Dheeraj Kalra & Mayank Srivastava

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Kalra, D., Goyal, V. & Srivastava, M. Design and performance analysis of low power LNA with variable gain current reuse technique. Analog Integr Circ Sig Process 108 , 351–361 (2021). https://doi.org/10.1007/s10470-021-01855-6

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DOI : https://doi.org/10.1007/s10470-021-01855-6

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What is LNA and Why is it Such a Powerful Research Tool?

LNA enhancement enables highly sensitive and specific analysis of short RNA and DNA targets.

LNA oligonucleotides have substantially increased affinity for their complementary strand, when compared with traditional DNA or RNA oligonucleotides. This results in unprecedented sensitivity and specificity and makes LNA-enhanced oligos excellent tools for detecting and differentiating small or highly similar DNA or RNA targets in many research applications.

Gene Expression, life science, female scientist working in a lab

  • What are locked nucleic acids (LNA)?
  • Why use LNA?

LNA has been successfully used to overcome the difficulties of studying very short sequences and has greatly improved, and in many cases enabled, specific and sensitive detection of non-coding RNA and other small RNA molecules. The unique ability of LNA oligonucleotides to discriminate between highly similar sequences has been further exploited in a number of applications targeting longer RNA sequences, such as mRNA. In addition, LNA has been successfully used for detection of low-abundance nucleic acids and chromosomal DNA.

The benefits of LNA include:

  • Significantly increased sensitivity compared to DNA and RNA oligos/probes
  • Robust detection of all miRNA sequences, regardless of GC content
  • Superior detection from challenging samples, such as biofluids and FFPE samples
  • Increased target specificity compared to DNA and RNA probes
  • Enables detection of single nucleotide mismatches
  • Superior discrimination of miRNA families
  • High in vivo and in vitro stability
  • Enables high potency binding to RNA and DNA
  • Superior antisense inhibition of small RNA targets in vivo

The affinity-enhancing effects of LNA give LNA oligonucleotides strand invasion properties, making LNA excellent for in vivo applications. Incorporation of LNA into oligonucleotides further increases resistance to endonucleases and exonucleases, which leads to high in vitro and in vivo stability. Since the physical properties (e.g., water solubility) of these sequences are very similar to those of RNA and DNA, conventional experimental protocols can easily be adjusted for their use.

  • Tm normalization – robust detection regardless of GC content
  • Superior single nucleotide discrimination
  • Superior results from challenging samples

The increase in sensitivity and specificity of LNA-enhanced oligonucleotides makes them ideal for challenging applications, in which the target is present at low levels.

For example, LNA-enhanced PCR primers are superior for quantifying short RNAs in small amounts of biofluids, such as serum and plasma (1), and LNA-enhanced capture probes offer excellent sensitivity and signal-to-noise ratios in FFPE samples, where short RNA targets, such as miRNAs, are present in a background of highly degraded RNA.

  • LNA in miRNA studies
  • Broad applicability across application areas

The unique characteristics of LNA make it a powerful tool, not only for miRNA research but also for detection of low-abundance, short or highly similar targets in a number of applications.

  • Discover our LNA-enhanced research tools

QIAGEN offers a diverse portfolio of high-performance LNA-enhanced tools to help you get to scientific insights more quickly. Find the right tool for your particular application using the links below:

  • Next-generation sequencing
  • RNA/miRNA functional analysis
  • RNA/miRNA localization studies
  • SNP detection
  • Jensen et al. (2011) Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs. BMC Genomics. 12:435. doi: 10.1186/1471-2164-12-435.
  • Open access
  • Published: 04 March 2015

An astute synthesis of locked nucleic acid monomers

  • Vivek K Sharma 1 ,
  • Pallavi Rungta 1 ,
  • Vipin K Maikhuri 1 &
  • Ashok K Prasad 1  

Sustainable Chemical Processes volume  3 , Article number:  2 ( 2015 ) Cite this article

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Novel attributes of Locked Nucleic Acid (LNA) makes it preferable over most of the other classes of modified nucleic acid analogues and therefore, it has been extensively explored in different synthetic oligonucleotide based therapeutics. In addition to five oligonucleotides of this class undergoing clinical trials, a healthy pipeline in pre-clinical studies validates the tenacity of LNA. Due to the increasing demand, an efficient biocatalytic methodology has recently been devised for the convergent synthesis of LNA monomers via selective enzymatic monoacetylation of diastereotopic hydroxymethyl functions of 3- O -benzyl-4- C -hydroxymethyl-1,2- O -isopropylidene- α -D-ribofuranose. This commentary article provides an insight into the different synthetic strategies followed for the synthesis of LNA monomers and their triumphs in clinical biotechnology.

Since the acclamation of nucleic acid therapeutics, modification in the sugar moiety of nucleosides has continuously reflected its supremacy for developing drug candidates for the treatment of cancer and viral infections [ 1 - 3 ]. After the pioneering development in dideoxy- and acyclic- nucleos(t)ides [ 3 ], currently the most promising modification in the ribofuranose moiety has appeared through the inclusion of an extra methylene bridge between 2′- O & 4′- C atom and synthesis of oligonucleotides (ONs) involving the modified nucleosides, termed as locked nucleic acid (LNA) (Figure  1 ) [ 4 , 5 ]. Although, no significant potency was observed against cancer or viral infections by LNA monomers or its analogues [ 6 ]; there is hardly any synthetic oligonucleotide (ON) based therapeutic strategy which has not been allured by their unique features [ 4 , 5 , 7 , 8 ].

Structure and conformations of DNA, RNA and LNA; B = nucleobase.

Seminal papers on LNA were independently instigated by Wengel [ 9 , 10 ] and Imanishi [ 11 ] groups. It is well known that the B-form DNA duplex possesses C 2′ - endo ( S -type) and the A-form RNA duplex has C 3′ - endo ( N -type) sugar puckering [ 12 , 13 ]. LNA is considered to be RNA mimic as the ancillary methylene bridge locks the sugar moiety into N -type sugar ring conformation (Figure  1 ). This conformational restriction results in preorganization of the backbone of LNA ONs, which leads to energetically favorable duplex formation via increased base stacking interactions according to standard Watson-Crick base pairing rules [ 14 ]. Generally, the melting temperature ( T m ) of duplexes is raised by 2-8°C per LNA nucleotide incorporation when compared to the corresponding unmodified duplexes, depending on the sequence context and number of modifications [ 14 - 16 ]. This makes LNA the prime nucleotide modification candidate for the applications where high hybridization affinity is desirable.

LNA-modified ONs have been extensively utilized in different approaches to target the corresponding nucleic acid counterparts. These primarily include, (a) antigene approach to block transcription of a particular gene; (b) antisense approach to induce RNA degradation; (c) siRNA mediated RNA degradation; and (d) blocking of microRNA [ 7 , 8 ]. Since LNA possess high hybridization affinity and target selectivity, it is unsurprising that increasing success in cell-line based experiments has paved their way to five LNA-based modified ONs under active clinical trials (Table  1 ). One of the most advanced LNA-based drugs Miravirsen which has entered Phase II clinical study is being developed by Santaris Pharma A/S. Miravirsen is an inhibitor of miR-122, a liver specific microRNA that is required by Hepatitis C virus (HCV) for replication. The liver-expressed miR-122 protects HCV from degradation. Miravirsen is designed to recognize and sequester miR-122, making it unavailable for HCV. As a result, the replication of the virus is effectively inhibited and the level of HCV is profoundly reduced (Figure  2 ) [ 17 ]. If its phase III trial looks anything like its phase II, Miravirsen could be the first LNA-based drug to get FDA approval.

Mechanism of action of chemically modified drug Miravirsen. (LNA monomers in oligonucleotide are shown red).

Two general strategies have been employed for the synthesis of LNA monomers; a linear strategy using commercially available RNA nucleosides as the starting material [ 11 , 19 ] and a convergent strategy where a common glycosyl donor is synthesized for coupling with different nucleobases [ 10 , 20 , 21 ]. Linear strategy was disclosed by Obika et al . [ 11 ] for the synthesis of LNA-U monomer 1a with uridine ( 2 ) as the starting material (Scheme  1 ).

Linear synthesis of LNA-U monomer [ 11 ]. Reagents (% yields ): (i) Cyclohexanone, PTSA (quantitative); (ii) 2-iodobenzoic acid, CH 3 CN (76%); (iii) (a) 37% HCHO, 2N NaOH, 1,4-dioxane; (b) NaBH 4 (38%); (iv) (a) TsCl, Pyridine; (b) TFA, water (34%); (v) PhCHO, ZnCl 2 , (80%); (vi) NaBH 3 CN, TiCl 4 , CH 3 CN (75%); (vii) NaHMDS, THF (61%); (viii) 10% Pd-C, H 2 , MeOH (quantitative).

Following similar strategy, Koshkin et al . [ 19 ] synthesized LNA-A monomer taking adenosine as the starting material. Despite having some advantages, such as cheap and readily available RNA nucleosides as starting material and short synthetic route to LNA monomers, the linear approach suffers from poor yields. The two key reactions in the synthetic pathway, i.e . the introduction of the additional hydroxymethyl group at the C -4′-position of the protected RNA nucleoside 4 and the regioselective tosylation of the introduced 4′- C -hydroxymethyl group, generally proceeds with very low yields (Scheme  1 ).

Alternatively, in the quest to establish a general method for the synthesis of all LNA monomers i.e . LNA-U, 1a ; LNA-T, 1b ; LNA-A, 1c and LNA-C, 1d ; the convergent strategy was explored by Koshkin et al . [ 10 ] using 3- O -benzyl-4- C -hydroxymethyl-1,2- O -isopropylidene- α -D-ribofuranose ( 10 ) as a starting material, which can be synthesized easily from D-glucose [ 22 , 23 ]. Regioselective 5- O -benzylation of 10 followed by acetolysis afforded the furanose 12 in 55% yield, a key intermediate for coupling reactions with a variety of nucleobases. The Vorbrüggen coupling with silylated nucleobases afforded the nucleosides 13a - d , which on deacetylation led to the formation of benzylated nucleosides 14a - d . The tosylation of the primary hydroxyl group in benzylated nucleosides 14a - d followed by in situ base-induced intramolecular ring closure afforded the 2′- O ,4′- C -linked locked nucleoside derivatives 15a - d . Debenzylation on dibenzylated nucleosides 15a - d efficiently yielded the LNA monomers 1a - d (Scheme  2 ).

Convergent synthesis of LNA monomers [ 10 ]. Reagents (% yields ): (i) BnBr, NaH, DMF (71%); (ii) (a) Ac 2 O, Pyridine; (b) 80% AcOH; (c) Ac 2 O, Pyridine (77%); (iii) nucleobase, N , O - bis (trimethylsilyl)acetamide, TMS-triflate, CH 3 CN or 1,2-dichloroethane ( 13a , 75%; 13b , 76%; 13c , 52%; 13d , 74%); (iv) NaOMe, MeOH ( 14a , 95%; 14b , 97%; 14c , 73%; 14d , 54%); (v) (a) TsCl, Pyridine; (b) NaH, DMF ( 15a , 30%; 15b , 42%; 15c , 44%; 15d , 51%); (vi) 20% Pd(OH) 2 -C, H 2 , EtOH ( 1a , 78%; 1b , 98%); BCl 3 , DCM ( 1c , 84%); 10% Pd-C, 1,4-cyclohexadiene, MeOH ( 1d , 36%).

Although, using the convergent strategy (Scheme  2 ), synthesis of LNA monomers with all natural nucleobases was standardized, regioselective benzylation of dihydroxy furanose derivative 10 remained unanswered [ 10 ]. Hence, in order to avoid the regioselective transformation on the furanose diol 10 , an alternate convergent synthesis was optimized by Koshkin et al . [ 20 ] (Scheme  3 ). Permesylation of furanose diol 10 afforded the dimesylated derivative 16 , which on acetolysis followed by acetylation, afforded an anomeric mixture of 1,2-di- O -acetyl-3- O -benzyl-4- C -methanesulfonyloxymethyl-5- O -methanesulfonyl-D-ribofuranose ( 17 ). The glycosyl donor 17 was used as a common intermediate for coupling reactions with different nucleobases to afford the LNA monomers 1a - d [ 20 , 21 ] as shown in Scheme  3 .

Improved convergent synthesis of LNA monomers [ 20 , 21 ]. Reagents (% yields ): (i) MsCl, pyridine, CH 2 Cl 2 (98%); (ii) Ac 2 O, AcOH, conc. H 2 SO 4 (97%); (iii) nucleobase, N , O - bis (trimethylsilyl)acetamide, TMS-triflate, CH 3 CN or 1,2-dichloroethane ( 18a , 90%; 18b , 88%; 18c , 68%; 18d , 82%); (iv) aq. NaOH, THF or dioxane ( 19a , 97%; 19b , 94%; 19c , 78%; 19d , 87%); (v) NaOBz, DMF ( 20a , 97%; 20b , 86%; 20c , 88%; 20d , 93%); (vi) aq. NaOH, THF ( 9a , 95%; 9b , 91%); NH 4 OH, MeOH ( 9c , 86%); (a) 20% Pd(OH) 2 -C, HCO 2 NH 4 , MeOH; (b) NH 4 OH ( 1d , 77%); (vii) 20% Pd(OH) 2 -C, 88% HCOOH, THF/MeOH (9:1) ( 1a , 91%); 20% Pd(OH) 2 -C, HCO 2 NH 4 , MeOH or EtOH ( 1b , 83%; 1c , 91%).

It seems easy to synthesize LNA monomers following the convergent strategy which utilizes the furanose diol 10 as the starting material. However, the use of 10 was found to be complicated due to the presence of two diastereotopic hydroxymethyl groups (Scheme  2 and Scheme  3 ). Therefore, we focused our attention towards lipase mediated diastereoselective protection of one of the hydroxymethyl groups in the crucial intermediate 10 with a base labile group such as acetyl, that can be hydrolyzed insitu concomitantly with 2′- O ,4′- C -cyclization towards the end of the synthesis to get the LNA monomers [ 24 ]. Screening of different lipases in organic solvents for the diasteroeselective acetylation of one of the two hydroxyl groups in dihydroxy compound 3- O -benzyl-4- C -hydroxymethyl-1,2- O -isopropylidene- α -D-ribofuranose ( 10 ) revealed that Candida antarctica lipase-B (Novozyme®-435) in diisopropyl ether (DIPE) in the presence of vinyl acetate as acetyl donor carries out the selective 5′- O -monoacetylation (Scheme  4 ).

Novozyme ® -435 mediated monoacetylation of diol 10 [ 24 ].

In a successful biocatalytic transformation reaction, a solution of the compound 10 and vinyl acetate in DIPE was incubated with Novozyme®-435 (10% w/w of 10 ) at 45°C and 200 rpm in an incubator shaker. The progress of the reaction was monitored on analytical TLC. On completion, reaction was quenched by filtering off the enzyme and solvent was removed under reduced pressure. The crude product thus obtained was washed with hexane to afford monoacetylated compound 5- O -acetyl-3- O -benzyl-4- C -hydroxymethyl-1,2- O -isopropylidene- α -D-ribofuranose ( 21 ) in quantitative yield. Using the optimized conditions, Novozyme®-435 was utilised for ten recycles of selective acetylation of compound 10 and was found to be equally regioselective for each cycle (Figure  3 ).

Study of recyclability of lipase Novozyme®-435 for acetylation reaction on diol 10. (All these reactions were performed on 100 mg batch size w.r.t. 10 ).

The synthesis of LNA monomers 1a - d was successfully achieved from enzyme-mediated monoacetylated compound 21 . Tosylation of 21 afforded compound 22 which on subsequent acetolysis gave an anomeric mixture 23 in 95% overall yield. Aiming for the convergent synthesis of LNA monomers, the mixture 23 was used as common glycosyl donor for the Vorbrüggen’s coupling reaction with uracil, thymine, 6- N -benzoyladenine and cytosine to yield the corresponding 2′,5′-di- O -acetyl-3′- O -benzyl-4′- C - p -toluenesulfonyloxymethyl-ribonucleosides 24a - d in 71-89% yields. Subsequently, deacetylation and concomitant intramolecular cyclization under alkaline conditions afforded the 3′- O -benzyl-2′- O ,4′- C -methylene-ribonucleosides 9a - d in 89-95% yields. Deprotection of the 3′- O -benzyl group in nucleosides 9a - d afforded the LNA monomers, i.e . 2′- O ,4′- C -methyleneuridine ( 1a ), 2′- O ,4′- C -methylenethymidine ( 1b ), 2′- O ,4′- C -methyleneadenosine ( 1c ) and 2′- O ,4′- C -methylenecytidine ( 1d ) in 81-91% yields (Scheme  5 ).

Chemo-enzymatic convergent synthesis of 2 '- O ,4 '- C - methylene - ribonucleosides (LNA monomers) 1a - d [ 24 ]. Reagents & conditions (% yields ): (i) TsCl, pyridine, CH 2 Cl 2 , 0°C to rt (98%); (ii) Ac 2 O, AcOH, H 2 SO 4 (100:10:0.1), 0°C to rt (97%); (iii) nucleobase, N , O - bis (trimethylsilyl) acetamide, TMS-triflate, acetonitrile or 1,2-dichloroethane, 80°C (24a, 89%; 24b, 88%; 24c, 71%; 24d, 78%); (iv) aq. NaOH, 1,4-dioxane, rt (+NH 4 OH for 9c) (9a, 95%; 9b, 93%; 9c, 89%; 9d, 94%); (v) 20% Pd(OH) 2 -C, HCOOH, THF:MeOH (9:1), reflux (1a, 91%); 20% Pd(OH) 2 -C, HCO 2 NH 4 , MeOH or EtOH, reflux (1b, 83%; 1c, 91%; 1d, 81%).

Starting from the diol 10 , the overall yields for developed chemo-enzymatic convergent synthesis of LNA monomers (Scheme  5 ) have been compared with the literature convergent methodology (Scheme  3 ). The results revealed that the developed biocatalytic methodology is more efficient in all cases with remarkable improvement for LNA-A (Table  2 ).

Unprecedented success of Locked Nucleic acid (LNA) in oligonucleotide based therapeutics demands a cost efficient, convenient and environment friendly synthetic route for LNA monomers. Therefore, Novozyme®-435 mediated selective protection of 3- O -benzyl-4- C -hydroxymethyl-1,2- O -isopropylidene- α -D-ribofuranose has been highlighted which lead to relatively efficient and environment friendly synthesis of LNA monomers in comparison to the earlier reports.

Abbreviations

  • Locked nucleic acid

Oligonucleotide

Hepatitis C virus

Diisopropyl ether

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Acknowledgments

We are grateful to the University of Delhi for providing financial support under DU-DST Purse Grant and under scheme to strengthen research and development. VKS and PR thank CSIR, and VKM thanks DBT, New Delhi for the award of JRF/SRF Fellowships.

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Sharma, V.K., Rungta, P., Maikhuri, V.K. et al. An astute synthesis of locked nucleic acid monomers. sustain chem process 3 , 2 (2015). https://doi.org/10.1186/s40508-015-0028-3

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  • Nucleic acid therapeutics
  • Bio-catalysis
  • Novozyme®-435
  • Modified oligonucleotides
  • Linear synthesis
  • Convergent synthesis

term paper on lna

LNA: a versatile tool for therapeutics and genomics

Affiliation.

  • 1 Nucleic Acid Center, Dept of Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark.
  • PMID: 12573856
  • DOI: 10.1016/S0167-7799(02)00038-0

Locked nucleic acid (LNA) is a nucleic acid analogue that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide-based therapeutics. For example, Tat-dependent transcription and telomerase activity have been efficiently suppressed by LNA oligomers, and efficient cleavage of highly structured RNA has been achieved using LNA-modified DNAzymes ('LNAzyme'). Furthermore, convincing examples of the application of LNA to nucleic acid diagnostics have been reported, including high capturing efficiencies and unambiguous scoring of single-nucleotide polymorphisms.

Publication types

  • Research Support, Non-U.S. Gov't
  • Biomimetic Materials / chemistry
  • Biomimetic Materials / metabolism
  • Biomimetic Materials / therapeutic use
  • DNA / chemistry
  • Gene Expression Profiling / methods
  • Gene Expression Regulation / drug effects
  • Gene Expression Regulation / genetics
  • Genetic Therapy / methods
  • Genomics / methods*
  • Hybridization, Genetic
  • In Situ Hybridization / methods*
  • Macromolecular Substances
  • Molecular Structure
  • Oligonucleotides
  • Oligonucleotides, Antisense / chemistry*
  • Oligonucleotides, Antisense / metabolism
  • Oligonucleotides, Antisense / therapeutic use*
  • Polymorphism, Single Nucleotide
  • RNA, Double-Stranded / chemistry
  • Oligonucleotides, Antisense
  • RNA, Double-Stranded
  • locked nucleic acid
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Everything You Need to Know to Write an A+ Term Paper

Last Updated: March 4, 2024 Fact Checked

Sample Term Papers

Researching & outlining.

  • Drafting Your Paper
  • Revising Your Paper

Expert Q&A

This article was co-authored by Matthew Snipp, PhD and by wikiHow staff writer, Raven Minyard, BA . C. Matthew Snipp is the Burnet C. and Mildred Finley Wohlford Professor of Humanities and Sciences in the Department of Sociology at Stanford University. He is also the Director for the Institute for Research in the Social Science’s Secure Data Center. He has been a Research Fellow at the U.S. Bureau of the Census and a Fellow at the Center for Advanced Study in the Behavioral Sciences. He has published 3 books and over 70 articles and book chapters on demography, economic development, poverty and unemployment. He is also currently serving on the National Institute of Child Health and Development’s Population Science Subcommittee. He holds a Ph.D. in Sociology from the University of Wisconsin—Madison. There are 13 references cited in this article, which can be found at the bottom of the page. This article has been fact-checked, ensuring the accuracy of any cited facts and confirming the authority of its sources. This article has been viewed 2,223,692 times.

A term paper is a written assignment given to students at the end of a course to gauge their understanding of the material. Term papers typically count for a good percentage of your overall grade, so of course, you’ll want to write the best paper possible. Luckily, we’ve got you covered. In this article, we’ll teach you everything you need to know to write an A+ term paper, from researching and outlining to drafting and revising.

Quick Steps to Write a Term Paper

  • Hook your readers with an interesting and informative intro paragraph. State your thesis and your main points.
  • Support your thesis by providing quotes and evidence that back your claim in your body paragraphs.
  • Summarize your main points and leave your readers with a thought-provoking question in your conclusion.

term paper on lna

  • Think of your term paper as the bridge between what you’ve learned in class and how you apply that knowledge to real-world topics.
  • For example, a history term paper may require you to explore the consequences of a significant historical event, like the Civil War. An environmental science class, on the other hand, may have you examine the effects of climate change on a certain region.
  • Your guidelines should tell you the paper’s word count and formatting style, like whether to use in-text citations or footnotes and whether to use single- or double-spacing. If these things aren’t specified, be sure to reach out to your instructor.

Step 2 Choose an interesting topic.

  • Make sure your topic isn’t too broad. For example, if you want to write about Shakespeare’s work, first narrow it down to a specific play, like Macbeth , then choose something even more specific like Lady Macbeth’s role in the plot.
  • If the topic is already chosen for you, explore unique angles that can set your content and information apart from the more obvious approaches many others will probably take. [3] X Research source
  • Try not to have a specific outcome in mind, as this will close you off to new ideas and avenues of thinking. Rather than trying to mold your research to fit your desired outcome, allow the outcome to reflect a genuine analysis of the discoveries you made. Ask yourself questions throughout the process and be open to having your beliefs challenged.
  • Reading other people's comments, opinions, and entries on a topic can often help you to refine your own, especially where they comment that "further research" is required or where they posit challenging questions but leave them unanswered.

Step 3 Do your research.

  • For example, if you’re writing a term paper about Macbeth , your primary source would be the play itself. Then, look for other research papers and analyses written by academics and scholars to understand how they interpret the text.

Step 4 Craft your thesis statement.

  • For example, if you’re writing a paper about Lady Macbeth, your thesis could be something like “Shakespeare’s characterization of Lady Macbeth reveals how desire for power can control someone’s life.”
  • Remember, your research and thesis development doesn’t stop here. As you continue working through both the research and writing, you may want to make changes that align with the ideas forming in your mind and the discoveries you continue to unearth.
  • On the other hand, don’t keep looking for new ideas and angles for fear of feeling confined. At some point, you’re going to have to say enough is enough and make your point. You may have other opportunities to explore these questions in future studies, but for now, remember your term paper has a finite word length and an approaching due date!

Step 5 Develop an outline for the paper.

  • Abstract: An abstract is a concise summary of your paper that informs readers of your topic, its significance, and the key points you’ll explore. It must stand on its own and make sense without referencing outside sources or your actual paper.
  • Introduction: The introduction establishes the main idea of your paper and directly states the thesis. Begin your introduction with an attention-grabbing sentence to intrigue your readers, and provide any necessary background information to establish your paper’s purpose and direction.
  • Body paragraphs: Each body paragraph focuses on a different argument supporting your thesis. List specific evidence from your sources to back up your arguments. Provide detailed information about your topic to enhance your readers’ understanding. In your outline, write down the main ideas for each body paragraph and any outstanding questions or points you’re not yet sure about.
  • Results: Depending on the type of term paper you’re writing, your results may be incorporated into your body paragraphs or conclusion. These are the insights that your research led you to. Here you can discuss how your perspective and understanding of your topic shifted throughout your writing process.
  • Conclusion: Your conclusion summarizes your argument and findings. You may restate your thesis and major points as you wrap up your paper.

Drafting Your Term Paper

Step 1 Make your point in the introduction.

  • Writing an introduction can be challenging, but don’t get too caught up on it. As you write the rest of your paper, your arguments might change and develop, so you’ll likely need to rewrite your intro at the end, anyway. Writing your intro is simply a means of getting started and you can always revise it later. [10] X Trustworthy Source PubMed Central Journal archive from the U.S. National Institutes of Health Go to source
  • Be sure to define any words your readers might not understand. For example, words like “globalization” have many different meanings depending on context, and it’s important to state which ones you’ll be using as part of your introductory paragraph.

Step 2 Persuade your readers with your body paragraphs.

  • Try to relate the subject of the essay (say, Plato’s Symposium ) to a tangentially related issue you happen to know something about (say, the growing trend of free-wheeling hookups in frat parties). Slowly bring the paragraph around to your actual subject and make a few generalizations about why this aspect of the book/subject is so fascinating and worthy of study (such as how different the expectations for physical intimacy were then compared to now).

Step 3 Summarize your argument with your conclusion.

  • You can also reflect on your own experience of researching and writing your term paper. Discuss how your understanding of your topic evolved and any unexpected findings you came across.

Step 4 Write your abstract.

  • While peppering quotes throughout your text is a good way to help make your point, don’t overdo it. If you use too many quotes, you’re basically allowing other authors to make the point and write the paper for you. When you do use a quote, be sure to explain why it is relevant in your own words.
  • Try to sort out your bibliography at the beginning of your writing process to avoid having a last-minute scramble. When you have all the information beforehand (like the source’s title, author, publication date, etc.), it’s easier to plug them into the correct format.

Step 6 Come up with a good title.

Revising & Finalizing Your Term Paper

Step 1 Make your writing as concise as possible.

  • Trade in weak “to-be” verbs for stronger “action” verbs. For example: “I was writing my term paper” becomes “I wrote my term paper.”

Step 2 Check for grammar and spelling errors.

  • It’s extremely important to proofread your term paper. If your writing is full of mistakes, your instructor will assume you didn’t put much effort into your paper. If you have too many errors, your message will be lost in the confusion of trying to understand what you’ve written.

Step 3 Have someone else read over your paper.

  • If you add or change information to make things clearer for your readers, it’s a good idea to look over your paper one more time to catch any new typos that may have come up in the process.

Matthew Snipp, PhD

  • The best essays are like grass court tennis—the argument should flow in a "rally" style, building persuasively to the conclusion. Thanks Helpful 0 Not Helpful 0
  • If you get stuck, consider giving your professor a visit. Whether you're still struggling for a thesis or you want to go over your conclusion, most instructors are delighted to help and they'll remember your initiative when grading time rolls around. Thanks Helpful 0 Not Helpful 0
  • At least 2 hours for 3-5 pages.
  • At least 4 hours for 8-10 pages.
  • At least 6 hours for 12-15 pages.
  • Double those hours if you haven't done any homework and you haven't attended class.
  • For papers that are primarily research-based, add about two hours to those times (although you'll need to know how to research quickly and effectively, beyond the purview of this brief guide).

term paper on lna

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  • ↑ https://www.binghamton.edu/counseling/self-help/term-paper.html
  • ↑ Matthew Snipp, PhD. Research Fellow, U.S. Bureau of the Census. Expert Interview. 26 March 2020.
  • ↑ https://emory.libanswers.com/faq/44525
  • ↑ https://writing.wisc.edu/handbook/assignments/planresearchpaper/
  • ↑ https://owl.purdue.edu/owl/general_writing/the_writing_process/thesis_statement_tips.html
  • ↑ https://libguides.usc.edu/writingguide/outline
  • ↑ https://gallaudet.edu/student-success/tutorial-center/english-center/writing/guide-to-writing-introductions-and-conclusions/
  • ↑ https://www.ncbi.nlm.nih.gov/pubmed/26731827
  • ↑ https://writing.wisc.edu/handbook/assignments/writing-an-abstract-for-your-research-paper/
  • ↑ https://www.ivcc.edu/stylesite/Essay_Title.pdf
  • ↑ https://www.uni-flensburg.de/fileadmin/content/institute/anglistik/dokumente/downloads/how-to-write-a-term-paper-daewes.pdf
  • ↑ https://library.sacredheart.edu/c.php?g=29803&p=185937
  • ↑ https://www.cornerstone.edu/blog-post/six-steps-to-really-edit-your-paper/

About This Article

Matthew Snipp, PhD

If you need to write a term paper, choose your topic, then start researching that topic. Use your research to craft a thesis statement which states the main idea of your paper, then organize all of your facts into an outline that supports your thesis. Once you start writing, state your thesis in the first paragraph, then use the body of the paper to present the points that support your argument. End the paper with a strong conclusion that restates your thesis. For tips on improving your term paper through active voice, read on! Did this summary help you? Yes No

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term paper on lna

How to Write a Term Paper From Start to Finish

term paper on lna

The term paper, often regarded as the culmination of a semester's hard work, is a rite of passage for students in pursuit of higher education. Here's an interesting fact to kick things off: Did you know that the term paper's origins can be traced back to ancient Greece, where scholars like Plato and Aristotle utilized written works to explore and document their philosophical musings? Just as these great minds once wrote their thoughts on parchment, you, too, can embark on this intellectual voyage with confidence and skill.

How to Write a Term Paper: Short Description

In this article, we'll delve into the core purpose of this kind of assignment – to showcase your understanding of a subject, your research abilities, and your capacity to communicate complex ideas effectively. But it doesn't stop there. We'll also guide you in the art of creating a well-structured term paper format, a roadmap that will not only keep you on track but also ensure your ideas flow seamlessly and logically. Packed with valuable tips on writing, organization, and time management, this resource promises to equip you with the tools needed to excel in your academic writing.

Understanding What Is a Term Paper

A term paper, a crucial component of your college education, is often assigned towards the conclusion of a semester. It's a vehicle through which educators gauge your comprehension of the course content. Imagine it as a bridge between what you've learned in class and your ability to apply that knowledge to real-world topics.

For instance, in a history course, you might be asked to delve into the causes and consequences of a significant historical event, such as World War II. In a psychology class, your term paper might explore the effects of stress on mental health, or in an environmental science course, you could analyze the impact of climate change on a specific region.

Writing a term paper isn't just about summarizing facts. It requires a blend of organization, deep research, and the art of presenting your findings in a way that's both clear and analytical. This means structuring your arguments logically, citing relevant sources, and critically evaluating the information you've gathered.

For further guidance, we've prepared an insightful guide for you authored by our expert essay writer . It's brimming with practical tips and valuable insights to help you stand out in this academic endeavor and earn the recognition you deserve.

How to Start a Term Paper

Before you start, keep the guidelines for the term paper format firmly in mind. If you have any doubts, don't hesitate to reach out to your instructor for clarification before you begin your research and writing process. And remember, procrastination is your worst enemy in this endeavor. If you're aiming to produce an exceptional piece and secure a top grade, it's essential to plan ahead and allocate dedicated time each day to work on it. Now, let our term paper writing services provide you with some valuable tips to help you on your journey:

start a term paper

  • Hone Your Topic : Start by cultivating a learning mindset that empowers you to effectively organize your thoughts. Discover how to research a topic in the section below.
  • Hook Your Readers: Initiate a brainstorming session and unleash a barrage of creative ideas to captivate your audience right from the outset. Pose intriguing questions, share compelling anecdotes, offer persuasive statistics, and more.
  • Craft a Concise Thesis Statement Example : If you find yourself struggling to encapsulate the main idea of your paper in just a sentence or two, it's time to revisit your initial topic and consider narrowing it down.
  • Understand Style Requirements: Your work must adhere to specific formatting guidelines. Delve into details about the APA format and other pertinent regulations in the section provided.
  • Delve Deeper with Research : Equipped with a clearer understanding of your objectives, dive into your subject matter with a discerning eye. Ensure that you draw from reputable and reliable sources.
  • Begin Writing: Don't obsess over perfection from the get-go. Just start writing, and don't worry about initial imperfections. You can always revise or remove those early sentences later. The key is to initiate the term papers as soon as you've amassed sufficient information.

Ace your term paper with EssayPro 's expert help. Our academic professionals are here to guide you through every step, ensuring your term paper is well-researched, structured, and written to the highest standards.

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Term Paper Topics

Selecting the right topic for your term paper is a critical step, one that can significantly impact your overall experience and the quality of your work. While instructors sometimes provide specific topics, there are instances when you have the freedom to choose your own. To guide you on how to write a term paper, consider the following factors when deciding on your dissertation topics :

choose a term paper topic

  • Relevance to Assignment Length: Begin by considering the required length of your paper. Whether it's a substantial 10-page paper or a more concise 5-page one, understanding the word count will help you determine the appropriate scope for your subject. This will inform whether your topic should be broad or more narrowly focused.
  • Availability of Resources : Investigate the resources at your disposal. Check your school or community library for books and materials that can support your research. Additionally, explore online sources to ensure you have access to a variety of reference materials.
  • Complexity and Clarity : Ensure you can effectively explain your chosen topic, regardless of how complex it may seem. If you encounter areas that are challenging to grasp fully, don't hesitate to seek guidance from experts or your professor. Clarity and understanding are key to producing a well-structured term paper.
  • Avoiding Overused Concepts : Refrain from choosing overly trendy or overused topics. Mainstream subjects often fail to captivate the interest of your readers or instructors, as they can lead to repetitive content. Instead, opt for a unique angle or approach that adds depth to your paper.
  • Manageability and Passion : While passion can drive your choice of topic, it's important to ensure that it is manageable within the given time frame and with the available resources. If necessary, consider scaling down a topic that remains intriguing and motivating to you, ensuring it aligns with your course objectives and personal interests.

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Term Paper Outline

Before embarking on the journey of writing a term paper, it's crucial to establish a well-structured outline. Be mindful of any specific formatting requirements your teacher may have in mind, as these will guide your outline's structure. Here's a basic format to help you get started:

  • Cover Page: Begin with a cover page featuring your name, course number, teacher's name, and the deadline date, centered at the top.
  • Abstract: Craft a concise summary of your work that informs readers about your paper's topic, its significance, and the key points you'll explore.
  • Introduction: Commence your term paper introduction with a clear and compelling statement of your chosen topic. Explain why it's relevant and outline your approach to addressing it.
  • Body: This section serves as the meat of academic papers, where you present the primary findings from your research. Provide detailed information about the topic to enhance the reader's understanding. Ensure you incorporate various viewpoints on the issue and conduct a thorough analysis of your research.
  • Results: Share the insights and conclusions that your research has led you to. Discuss any shifts in your perspective or understanding that have occurred during the course of your project.
  • Discussion: Conclude your term paper with a comprehensive summary of the topic and your findings. You can wrap up with a thought-provoking question or encourage readers to explore the subject further through their own research.

How to Write a Term Paper with 5 Steps

Before you begin your term paper, it's crucial to understand what a term paper proposal entails. This proposal serves as your way to introduce and justify your chosen topic to your instructor, and it must gain approval before you start writing the actual paper.

In your proposal, include recent studies or research related to your topic, along with proper references. Clearly explain the topic's relevance to your course, outline your objectives, and organize your ideas effectively. This helps your instructor grasp your term paper's direction. If needed, you can also seek assistance from our expert writers and buy term paper .

how to write a term paper

Draft the Abstract

The abstract is a critical element while writing a term paper, and it plays a crucial role in piquing the reader's interest. To create a captivating abstract, consider these key points from our dissertation writing service :

  • Conciseness: Keep it short and to the point, around 150-250 words. No need for lengthy explanations.
  • Highlight Key Elements: Summarize the problem you're addressing, your research methods, and primary findings or conclusions. For instance, if your paper discusses the impact of social media on mental health, mention your research methods and significant findings.
  • Engagement: Make your abstract engaging. Use language that draws readers in. For example, if your paper explores the effects of artificial intelligence on the job market, you might begin with a question like, 'Is AI revolutionizing our work landscape, or should we prepare for the robots to take over?'
  • Clarity: Avoid excessive jargon or technical terms to ensure accessibility to a wider audience.

Craft the Introduction

The introduction sets the stage for your entire term paper and should engage readers from the outset. To craft an intriguing introduction, consider these tips:

  • Hook Your Audience: Start with a captivating hook, such as a thought-provoking question or a compelling statistic. For example, if your paper explores the impact of smartphone addiction, you could begin with, 'Can you remember the last time you went a whole day without checking your phone?'
  • State Your Purpose: Clearly state the purpose of your paper and its relevance. If your term paper is about renewable energy's role in combating climate change, explain why this topic is essential in today's world.
  • Provide a Roadmap: Briefly outline how your paper is structured. For instance, if your paper discusses the benefits of mindfulness meditation, mention that you will explore its effects on stress reduction, emotional well-being, and cognitive performance.
  • Thesis Statement: Conclude your introduction with a concise thesis statement that encapsulates the central argument or message of your paper. In the case of a term paper on the impact of online education, your thesis might be: 'Online education is revolutionizing learning by providing accessibility, flexibility, and innovative teaching methods.'

Develop the Body Sections: Brainstorming Concepts and Content

Generate ideas and compose text: body sections.

The body of your term paper is where you present your research, arguments, and analysis. To generate ideas and write engaging text in the body sections, consider these strategies from our research paper writer :

  • Structure Your Ideas: Organize your paper into sections or paragraphs, each addressing a specific aspect of your topic. For example, if your term paper explores the impact of social media on interpersonal relationships, you might have sections on communication patterns, privacy concerns, and emotional well-being.
  • Support with Evidence: Back up your arguments with credible evidence, such as data, research findings, or expert opinions. For instance, when discussing the effects of social media on mental health, you can include statistics on social media usage and its correlation with anxiety or depression.
  • Offer Diverse Perspectives: Acknowledge and explore various viewpoints on the topic. When writing about the pros and cons of genetic engineering, present both the potential benefits, like disease prevention, and the ethical concerns associated with altering human genetics.
  • Use Engaging Examples: Incorporate real-life examples to illustrate your points. If your paper discusses the consequences of climate change, share specific instances of extreme weather events or environmental degradation to make the topic relatable.
  • Ask Thought-Provoking Questions: Integrate questions throughout your text to engage readers and stimulate critical thinking. In a term paper on the future of artificial intelligence, you might ask, 'How will AI impact job markets and the concept of work in the coming years?'

Formulate the Conclusion

The conclusion section should provide a satisfying wrap-up of your arguments and insights. To craft a compelling term paper example conclusion, follow these steps:

  • Revisit Your Thesis: Begin by restating your thesis statement. This reinforces the central message of your paper. For example, if your thesis is about the importance of biodiversity conservation, reiterate that biodiversity is crucial for ecological balance and human well-being.
  • Summarize Key Points: Briefly recap the main points you've discussed in the body of your paper. For instance, if you've been exploring the impact of globalization on local economies, summarize the effects on industries, job markets, and cultural diversity.
  • Emphasize Your Main Argument: Reaffirm the significance of your thesis and the overall message of your paper. Discuss why your findings are important or relevant in a broader context. If your term paper discusses the advantages of renewable energy, underscore its potential to combat climate change and reduce our reliance on fossil fuels.
  • Offer a Thoughtful Reflection: Share your own reflections or insights about the topic. How has your understanding evolved during your research? Have you uncovered any unexpected findings or implications? If your paper discusses the future of space exploration, consider what it means for humanity's quest to explore the cosmos.
  • End with Impact: Conclude your term paper with a powerful closing statement. You can leave the reader with a thought-provoking question, a call to action, or a reflection on the broader implications of your topic. For instance, if your paper is about the ethics of artificial intelligence, you could finish by asking, 'As AI continues to advance, what ethical considerations will guide our choices and decisions?'

Edit and Enhance the Initial Draft

After completing your initial draft, the revision and polishing phase is essential for improving your paper. Here's how to refine your work efficiently:

  • Take a Break: Step back and return to your paper with a fresh perspective.
  • Structure Check: Ensure your paper flows logically and transitions smoothly from the introduction to the conclusion.
  • Clarity and Conciseness: Trim excess words for clarity and precision.
  • Grammar and Style: Proofread for errors and ensure consistent style.
  • Citations and References: Double-check your citations and reference list.
  • Peer Review: Seek feedback from peers or professors for valuable insights.
  • Enhance Intro and Conclusion: Make your introduction and conclusion engaging and impactful.
  • Coherence Check: Ensure your arguments support your thesis consistently.
  • Read Aloud: Reading your paper aloud helps identify issues.
  • Final Proofread: Perform a thorough proofread to catch any remaining errors.

Term Paper Format

When formatting your term paper, consider its length and the required citation style, which depends on your research topic. Proper referencing is crucial to avoid plagiarism in academic writing. Common citation styles include APA and MLA.

If unsure how to cite term paper for social sciences, use the APA format, including the author's name, book title, publication year, publisher, and location when citing a book.

For liberal arts and humanities, MLA is common, requiring the publication name, date, and location for referencing.

Adhering to the appropriate term paper format and citation style ensures an organized and academically sound paper. Follow your instructor's guidelines for a polished and successful paper.

Term Paper Example

To access our term paper example, simply click the button below.

The timeline of events from 1776 to 1861, that, in the end, prompted the American Civil War, describes and relates to a number of subjects modern historians acknowledge as the origins and causes of the Civil War. In fact, pre-Civil War events had both long-term and short-term influences on the War—such as the election of Abraham Lincoln as the American president in 1860 that led to the Fall of Fort Sumter in April of the same year. In that period, contentions that surrounded states’ rights progressively exploded in Congress—since they were the initial events that formed after independence. Congress focused on resolving significant issues that affected the states, which led to further issues. In that order, the US’s history from 1776 to 1861 provides a rich history, as politicians brought forth dissimilarities, dissections, and tensions between the Southern US & the people of slave states, and the Northern states that were loyal to the Union. The events that unfolded from the period of 1776 to 1861 involved a series of issues because they promoted the great sectional crisis that led to political divisions and the build-up to the Civil War that made the North and the South seem like distinctive and timeless regions that predated the crisis itself.

Final Thoughts

In closing, approach the task of writing term papers with determination and a positive outlook. Begin well in advance, maintain organization, and have faith in your capabilities. Don't hesitate to seek assistance if required, and express your individual perspective with confidence. You're more than capable of succeeding in this endeavor!

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Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

Makoto ikenaga.

1 Research Field in Agriculture, Agriculture Fisheries and Veterinary Medicine Area, Kagoshima University, 1–21–24, Korimoto, Kagoshima, 890–0065, Japan

Masakazu Tabuchi

2 Graduate School of Agriculture, Kagoshima University, 1–21–24, Korimoto, Kagoshima, 890–0065, Japan

Tomohiro Kawauchi

3 The United Graduate School of Agricultural Sciences, Kagoshima University, 1–21–24, Korimoto, Kagoshima, 890–0065, Japan

Masao Sakai

Associated data.

The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi.

Plant–associated fungi promote the growth of host plants. Although some fungi have neutral or deleterious effects on plant growth, beneficial fungi protect plants by enhancing resistance to disease and insects ( 7 , 19 , 24 ), solubilizing insoluble soil phosphate ( 13 , 46 ), supplying available forms of nitrogen ( 32 ), and conferring heavy metal and drought tolerance ( 3 , 9 ). Therefore, investigations on plant–associated fungal community structures provide basic information that facilitates the isolation of beneficial fungi for agricultural applications as microbial materials or bio–fertilizers.

Cultivation methods have been used to characterize fungal communities ( 11 ). However, some unisolated and unexploited fungi remain in ecosystems, including those affiliated with plants ( 12 , 14 ). Hawksworth et al. ( 16 ) reported that 74K to 120K fungi have been identified to date, whereas 1.5M species were estimated as the total fungal population, indicating that more than 90% of fungal species are still unknown. Fungi that form large colonies on agar plates are preferentially detected in culture–dependent community analyses, resulting in the underestimation of other fungal community components ( 12 ). However, a culture–independent molecular technique is more advantageous for studying fungal communities because it provides information on differences between community structures irrespective of culturable, unisolated, and unexploited fungi. This approach has accelerated community analyses of plant–associated fungi.

In community analyses using culture–independent molecular techniques, small subunit (SSU) and large subunit (LSU) rRNA genes and the internal transcribed spacer (ITS) region are frequently used as barcoding sequences ( 17 , 30 , 42 , 43 , 49 , 59 ). The ITS region has been identified as a suitable barcoding region for a fungal community analysis because its sequences vary more than those of SSU and LSU rRNA genes ( 45 , 47 ). However, previous community analyses of plant–associated fungi have frequently been limited due to contamination by host plant DNA ( 15 , 18 ). This has been attributed to the regions of the SSU and LSU rRNA genes used in the design of primer sets to amplify the fungal ITS region, a sequence that is almost homologous to those found in host plant DNA ( 51 ), and results in an abundance of host plant amplicons in PCR products. This unspecific amplification has delayed understanding of the roles of fungi in plant growth. In response to this major limitation, fungal-specific primers were designed to amplify the ITS regions ( 15 , 35 ). However, these primers had low coverage for fungi, resulting in a PCR amplification bias ( 6 , 51 ).

A locked nucleic acid (LNA) is an artificial nucleotide analog that contains a methylene bridge connecting the 2′–oxygen of ribose with the 4′–carbon ( 26 , 39 ). This bridge results in a locked 3′– endo conformation with reduced conformational flexibility ( 29 , 33 ). The LNA base may be incorporated into oligonucleotide sequences similar to a DNA base ( 8 , 25 ). The incorporated LNA oligonucleotide (LNA oligonucleotide) shows extraordinary mismatch sensitivity to complementary nucleic acids in LNA/DNA hybrids ( 26 , 29 ), and higher thermal stability than DNA oligonucleotides ( 48 , 55 ).

We have developed an LNA oligonucleotide–PCR clamping technique in order to investigate the community structures of plant–associated bacteria ( 20 , 21 , 22 ). This technique has enabled the PCR amplification of bacterial SSU rRNA genes, while inhibiting the amplification of host plant organelle (mitochondria and plastid) genes. Although this innovative technique is considered to be applicable to the selective amplification of fungal ITS regions, it is still limited because the ITS regions of other DNA derived from microeukaryotes, such as protozoa, as well as those of the host plant are also amplified due to mismatches in fungal-specific primers during PCR ( 15 , 35 ). The PCR clamping technique may be employed when DNA sequences expected to be inhibited are already known; however, this technique is insufficient when DNA sequences are unspecified, such as in eukaryotic communities. In this case, the application of a fungal-specific primer incorporating LNA bases (an LNA primer) is suitable for avoiding mismatches in primer annealing for the selective amplification of fungal ITS regions on the forward and reverse sides. Alternatively, the combination of LNA primers and the LNA oligonucleotide–PCR clamping technique, in which LNA oligonucleotides are specific for host plant DNA predominantly contaminating extracted DNA, appears to be effective.

In order to develop this new method for a fungal community analysis, we selected wheat, soybean, and potato as representative plants, and targeted Ascomycota and Basidiomycota , which account for approximately 80% of all fungus species currently identified ( 6 ), together with the known arbuscular mycorrhizas, Glomeromycota . We then tested the effectiveness of the LNA technique in a community analysis of plant–associated fungi by comparing the DGGE patterns generated with and without the LNA technique. We also examined possible interference by LNA oligonucleotides during the PCR amplification of fungal ITS regions using DNA extracted from agricultural field soil.

Materials and Methods

Preparation of plant samples.

Wheat ( Triticum aestivum ‘Chikugoizumi’), soybean ( Glycine max ‘Fukuyutaka’), and potato ( Solanum tuberosum ‘Nishiyutaka’) were cultivated under upland conditions. In brief, brown lowland soil was collected from the plow layer of an agricultural field at Kagoshima University (latitude 31°34′ N, longitude 130°32′ E), and placed into 1/5000a pots. After germination, wheat and soybean were cultivated in the potted soils for several days. Twenty wheat and soybean seedlings were harvested, and the roots were collected separately. Potato seed tubers were directly transplanted into the pots and cultivated for one month. The leaves, stems, roots, and epidermises from secondary young tubers were separately collected from five individuals. The epidermises of mother tubers were also collected, and all samples were ground for DNA extraction and adjusted to 0.5 g fresh weight mL −1 with sterilized water.

In order to prepare control DNAs, which only produce amplicons of host plant ITS regions after PCR, the aseptic roots of wheat and soybean were prepared according to the protocol specified by Sakai and Ikenaga ( 44 ) using 1/10 tryptic soy agar ( 34 ). A small section of the inner tuber of the potato was aseptically collected. These samples were ground and adjusted following the same procedure, and regarded as aseptic samples.

DNA extraction from plant samples and soils

Approximately 0.4 g (ground and adjusted weight) of roots from wheat and soybean, and the leaves, stems, roots, and epidermises of young and mother tubers from potato were used for DNA extraction in duplicate by ISOPLANT II (Nippon Gene, Toyama, Japan) with the bead–beating method. The same amounts of aseptic samples prepared from wheat, soybean, and potato were used in the extraction. In brief, samples were physically shaken by bead–beating at 4,800 rpm for 60 s using MS–100 TOMY (TOMY, Tokyo, Japan), then chemically treated with benzyl chloride. Subsequent procedures followed the manufacturer’s instructions, with minor modifications.

Approximately 0.5 g fresh soil collected from the agricultural field in Kagoshima University was used for DNA extraction. Extraction was performed using the FastDNA SPIN Kit for Soil (MP Biomedicals, Solon OH, USA) according to the manufacturer’s instructions. Following the extraction of plant and soil samples, crude DNA was purified using the Power Clean DNA Clean UP Kit (MO BIO, Carlsbad, CA, USA).

Designing LNA primers and LNA oligonucleotides

The fungal and plant (wheat, soybean and potato) sequences of SSU rRNA and LSU rRNA genes, located upstream and downstream of the ITS region, respectively, were obtained from the nucleotide database of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov ). Fungal sequences were collected from the three major phyla Ascomycota , Basidiomycota , and Glomeromycota , which contain the sequences of plant–associated fungi.

Sequences were aligned with the primers used to amplify fungal ITS regions by CLUSTAL W version 1.7 ( 50 ), and the relevant sequences were organized by removing gaps using BioEdit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ). The possible sequences to design LNA primers specific to fungi or LNA oligonucleotides specific to plants for application to the clamping technique were examined in consideration of primer coverage.

Based on the aligned sequences, LNA primers were designed by converting the fungal-specific DNA base into an LNA base in order to enhance its specificity to fungi. LNA oligonucleotides were designed by overlapping a few DNA bases with the annealing position of the fungal primer at the extension side, and converting DNA bases, which were only specific to plants, into LNA bases at the shifted position. The 3′ ends of LNA oligonucleotides were phosphorylated to avoid extension during PCR. The sequences of LNA oligonucleotides were verified using the BLAST search program ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) ( 1 ) to confirm if identical sequences existed in fungi.

Estimation of the Tm values of LNA oligonucleotides

In order to estimate the Tm values of LNA oligonucleotides, PCR amplification was performed at different annealing temperatures using a PCR thermal cycler (PC350, ASTEC, Fukuoka, Japan). DNA extracted from wheat and soybean roots was used as representative samples. The PCR mixture contained Premix Hot Start Version (Takara, Otsu, Japan), the fungal primer set (0.8 μM each), and the DNA template. PCR conditions were as follows: 94°C for 3 min (initial denaturation), followed by 40 cycles at 94°C for 1 min, annealing from 60°C to 74°C at 2°C intervals for 1 min, and 72°C for 2 min, with a final extension step at 72°C for 10 min. Aliquots of the PCR products were electrophoresed after amplification. The Tm values of the LNA oligonucleotides were estimated from amplicon intensities.

Estimation of effective concentrations of LNA oligonucleotides

The extracted DNA of the respective samples was used in the amplification to estimate the effective concentrations of LNA oligonucleotides. The PCR mixtures contained serial concentrations of LNA oligonucleotides, and comprised 0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, and 4.0 μM. Mixtures of 6.0 μM and 8.0 μM were also prepared for potato leaf and stem samples. PCR tubes contained extracted DNA, forward and reverse primers (0.8 μM each as the final concentration), premix Ex Taq Hot Start Version (Takara), an LNA oligonucleotide (0 μM to 8.0 μM as the final concentration), and sterilized ultra-pure water. An LNA primer or DNA primer was used as a forward primer. PCR tubes, containing extracted DNA from the respective samples and aseptic samples, were also prepared without using the LNA technique.

In order to apply PCR clamping by LNA oligonucleotides, the annealing step for the LNA oligonucleotides was added between the denaturation step and annealing step of the fungal primers during the amplification process. A 1-min incubation period was added between the steps. The program implemented the following steps: 94°C for 3 min (initial denaturation), followed by 40 cycles at 94°C for 1 min, annealing of the LNA oligonucleotide for 1 min, 54°C for 1 min (annealing step of fungal primers) and 72°C for 2 min, with a final extension step at 72°C for 10 min. Aliquots of the PCR products were electrophoresed, and the effective concentrations of the LNA oligonucleotides were estimated by confirming the product trend, which exhibited the same mobility as the amplicons of aseptic samples.

Nested PCR amplification and DGGE

The PCR products obtained under optimized conditions were again amplified using the fungal primer set for denaturant gradient gel electrophoresis (DGGE). Prior to nested PCR, the products were purified and serially diluted 10 3 - to 10 4 -fold. The products amplified without the LNA technique were also prepared to compare DGGE patterns. In addition, extracted DNA from aseptic roots was directly amplified with the primer set used for DGGE in order to confirm the DGGE banding positions of host plants.

DGGE was performed in a gel containing a linear chemical gradient ranging from 20% to 60% of the denaturant ( 37 ). Approximately 600 ng of amplicons were loaded and electrophoresed at 60°C and 100 V for 14 h using a DCode universal mutation detection system (BioRad Laboratories, Hercules CA, USA). After electrophoresis, the gel was stained with SYBR gold (Life Technologies Japan, Tokyo, Japan) and photographed under UV illumination.

Sequencing of DGGE bands

The sequences of some DGGE bands, which were newly detected using the LNA technique, were obtained by direct sequencing or TA cloning–assisted sequencing.

In direct sequencing, the bands were excised from the gel and directly amplified as a DNA template. A mobility check of the amplified band was performed to confirm whether the position of the band was the same as that of the original by replicating the DGGE analysis under identical conditions. A forward primer without the GC clamp and a reverse primer for DGGE were used for cycle sequencing. DNA sequencing was performed using the ABI 3500 xL Genetic Analyzer (Life Technologies Japan).

In TA cloning–assisted sequencing, the amplicons used for DGGE were purified and ligated to the pT7 Blue T–vector (Novagen, Madison, WI, USA) with TA–Blunt Ligation Kit (Nippon Gene). The ligated products (plasmids) were transformed in competent cells of ECOS Competent E. coli XL1–Blue (Nippon Gene). After blue/white selection, the DGGE bands incorporated as inserts in the plasmid were amplified with the T7 primer (5′–TAATACGACTCA CTATAGG–3′) and U–19 primer (5′–GTTTTCCCAGTCACGAC GT–3′), and nested PCR was performed for the purified products with the primer set used for DGGE. A mobility check of the products was performed by DGGE in order to confirm the positions of the bands. The T7 and U–19 primers used for cycle sequencing corresponded to the purified T7 and U–19 products

Examination of possible interference by LNA oligonucleotides during PCR for the amplification of fungal DNA

The extracted DNA from agricultural field soil was amplified to examine the possible interference of LNA oligonucleotides in the amplification of fungal ITS regions during PCR. A reaction without LNA oligonucleotides was also conducted as a control. DGGE was performed in an acrylamide gel containing a linear chemical gradient ranging between 20% and 60%. The patterns generated with and without LNA oligonucleotides were compared.

Nucleotide sequence accession number

The sequences obtained in this study are available on DNA databases under the accession numbers LC0262298– {"type":"entrez-nucleotide","attrs":{"text":"LC026315","term_id":"764014729","term_text":"LC026315"}} LC026315 .

Results and Discussion

Investigation of designable regions for lna primers and lna oligonucleotides.

The forward and reverse primers used in the fungal community analysis were collected in order to identify designable regions for LNA primers and LNA oligonucleotides. The forward primers were ITS9mun ( 10 ), NSI1 ( 36 ), ITS1F ( 15 ), ITS1F_KYO1 and ITS1F_KYO2 ( 51 ), ITS5 ( 57 ), and ITS1 ( 57 ). The reverse primers were ITS4 ( 57 ), ITS4_KYO1, ITS4_KYO2, and ITS4_KYO3 ( 51 ), ITS8mun ( 10 ), NLB4 ( 36 ), and NLC3 ( 36 ). The forward and reverse primers were designed to hybridize downstream of 18S rRNA genes and upstream of 26S rRNA genes, respectively, in order to amplify fungal ITS regions for the community analysis.

Alignment was performed using the sequences of fungi derived from Ascomycota , Basidiomycota , and Glomeromycota , and of agricultural plants including wheat, soybean, and potato, together with primer sequences. After alignment on the forward side, LNA primers were only designed for the annealing positions of the NSI1 and ITS1F primers, while the positions to design LNA oligonucleotides for PCR clamping were not observed on the forward side (data not shown). When comparing fungal sequence coverage, the NSI1 primer had 77.9% coverage in the phyla Ascomycota , Basidiomycota , and Glomeromycota , while higher coverage (89.8%) was achieved by the ITS1F primer ( 51 ). In addition, the ITS1F primer was designed to contain specific bases to enhance the selective amplification of fungal DNA ( 15 ). These results suggest that the ITS1F primer is optimal for designing LNA primers by replacing specific bases with LNA bases.

On the other hand, the positions to design LNA primers were not observed on the reverse side (data not shown). However, a modifiable position for LNA oligonucleotides was found to be in competition with the ITS4 primer. Although the ITS4 primer has been designed to cover whole eukaryotes ( 57 ), specific bases for plants were found in a position that was shifted toward the extension side from the annealing position. Host plant DNA was generally abundant in extracts from plant samples. It is reasonable to inhibit its amplification by applying the LNA oligonucleotide–PCR clamping technique. As a result, the combination of an LNA primer on the forward side and the clamping technique on the reverse side was considered to be effective for enhancing the PCR amplification of the fungal ITS region. The primer set ITS1F and ITS4 has also frequently been used to amplify the fungal ITS region in environmental samples ( 2 , 5 , 27 , 41 , 56 ).

Designing LNA primers

Fig. 1 shows the alignment sequences of fungi and representative agricultural plants, including wheat, soybean and potato, on the forward side together with the ITS1F primer. The numbers of fungal sequences used in the alignment were 1,519, 1,483, and 317 as derived from Ascomycota , Basidiomycota , and Glomeromycota , respectively. These were not the numbers of species, but those of fungal sequences that were widely collected in consideration of phylogenetic diversities.

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The 3rd and 19th bases from the 5′ end were mostly T and G for the three fungal phyla. However, C was found at a certain percentage in both bases in Ascomycota . In order to increase the coverage of the ITS1F primer, these bases were degenerated as Y (T or C) and S (C or G) in the 3rd and 19th bases, respectively. The modified primer was named the ITS1F KU DNA primer, and its sequence was 5′–CTYGGTCATTTAGAGGAASTAA–3′.

In addition, the 5th, 20th, and 22nd bases from the 5′ end were G, T, and A in fungal sequences, while the corresponding bases were A, C, and G in plant sequences, indicating the availability of DNA bases to be converted to LNA bases in a fungal–specific LNA primer design. The LNA primer designed was named the ITS1F KU LNA primer, and its sequence was 5′–CTYG G TCATTTAGAGGAAS T A A –3′ (underlined bases indicated the LNA bases).

Estimation of Tm values for LNA oligonucleotides

In order to apply the PCR clamping technique, the annealing step of LNA oligonucleotides needs to be added between the denaturation step and annealing step of fungal primers, which inhibits the amplification of host plant DNA by LNA oligonucleotides. In this step, the annealing temperature for LNA oligonucleotides needs to be higher than that for the fungal primer, at which fungal primers are non–functional, thereby avoiding hybridization with host plant DNA. If fungal primers hybridize at the higher temperature, host plant DNA is easily amplified during PCR for excessive inclusion in DNA extracts. In addition, the annealing temperature for LNA oligonucleotides needs to be as low as possible. If the temperature is too high, the efficiency of hybridization for LNA oligonucleotides is decreased, resulting in the amplification of host plant DNA in the subsequent step. In order to avoid the hybridization of the ITS4 primer at the corresponding position of host plant DNA during the annealing step of LNA oligonucleotides, the Tm values of LNA oligonucleotides were estimated by PCR using the ITS1F KU LNA and ITS4 primers.

As shown in Fig. 2 , the amplification products of DNA extracted from wheat and soybean roots were detected at high intensities at 60°C. However, their intensities decreased until 64°C with an increase in the annealing temperature, and no products were detected at temperatures higher than 66°C. This result indicated that the primer set was functional up to 64°C; however, this temperature appeared to be high because the Tm values of the ITS1F KU LNA and ITS4 primers were 59–62°C and 60°C, respectively. Therefore, the high temperature resulted in low product intensities after PCR amplification. Based on the results of electrophoresis, the Tm values of LNA oligonucleotides were estimated to be approximately 70°C in order to avoid the annealing of fungal primers.

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Estimation of Tm values for LNA oligonucleotides. The annealing temperatures at which fungal primers were non–functional were used for the Tm values of LNA oligonucleotides. Annealing temperatures ranged between 60°C and 72°C, with increments of 2°C. “M” is the marker for the 100-bp ladders.

Designing ITS4 LNA oligonucleotides

Fig. 3 shows the alignment sequences of representative agricultural plants, including wheat, soybean, and potato, and fungi from three phyla on the reverse side to design LNA oligonucleotides that compete with the ITS4 primer at the annealing position. In this design, the numbers of fungal sequences used in the alignment were 1,289, 683, and 96, derived from Ascomycota , Basidiomycota , and Glomeromycota , respectively. These were also the numbers of fungal sequences widely collected in consideration of diversities.

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After alignment, wheat, soybean, and potato each had individual sequences in the LNA oligonucleotide position. The other representative plants were grouped into one of the three types: a) rice, maize, melon, and carrot, b) spinach, peanut, and banana and c) thale cress, tomato, sweet potato, coffee, orange, cotton, and jute. These groups showed the same sequences as wheat, soybean, and potato, respectively. The sequence types of other agricultural plants are listed in Supplementary Table S1 . Based on the alignment in Fig. 3 , three LNA oligonucleotides specific for wheat (type a), soybean (type b), and potato (type c) were designed by overlapping one or two bases with the 3′ end of the ITS4 primer, and by replacing specific bases for plants with LNA bases in order to equally distribute LNA bases throughout the sequences, with the exception of the overlapped base with the ITS4 primer. Simultaneously, Tm values were regulated at approximately 70°C using the automatic calculator on the EXIQON website ( https://www.exiqon.com/ls ). The LNA oligonucleotides designed were named ITS4 LNA oligonucleotides a, b, and c, respectively. The Tm values of ITS4 LNA oligonucleotides a, b, and c were 68°C, 72°C, and 69°C, and their sequences were 5′– C TTAA AC TCAGCGGGTAGTCC C p–3′, 5′– C TTAA AC TCAGCGGGTAG C CC C p–3′, and 5′– GC TT AA AC TCAGCGGGTA A TCC C p–3′, respectively. The bold bases indicate the bases overlapping with the ITS4 primer, and the LNA bases are underlined. The 3′ end was phosphorylated (indicated as p) to avoid extension from the oligonucleotides during PCR.

The BLAST search program was used to investigate whether the three oligonucleotides were identical to fungal sequences. LNA oligonucleotides a, b, and c completely matched 7, 1, and 6 fungal sequences, respectively. They were mostly affiliated with uncultured fungal clones. Owing to this negligible amount, the LNA oligonucleotides designed were considered to be available for PCR clamping in order to inhibit the amplification of host plant DNA.

Effective concentrations of the LNA oligonucleotides were examined using 0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, and 4.0 μM to extracted DNA from the respective plant samples ( Fig. 4 ). Higher concentrations of oligonucleotides, namely, 6.0 μM and 8.0 μM, were also examined with those of potato leaf and stem samples. ITS1F KU LNA and ITS4 primers were used as forward and reverse primers, respectively. In addition, two different amplicons were prepared; one was the product amplified using ITS1F KU DNA and ITS4 primers using DNA extracted from aseptic samples (Lane C). One was used to confirm the mobility locations of host plant products in agarose and DGGE gels, while the other, which was shown as LNA(−), was the product amplified with the ITS1F KU DNA and ITS4 primers using DNA extracted from the respective samples to produce amplicons prepared without LNA primers or PCR clamping by LNA oligonucleotides.

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Estimation of effective concentrations for LNA oligonucleotides. LNA oligonucleotides were used in the ranges of 0 μM, 0.5 μM, 1.0 μM, 2.0 μM, 3.0 μM, and 4.0 μM. Higher concentrations of 6.0 μM and 8.0 μM were also examined for potato leaf and stem samples. “M” is the marker for 100-bp ladders, and “C” is aseptic amplicons to provide the position of host plant DNA in the agarose gel. “LNA(−)” represents amplicons prepared with the ITS1F DNA primer and ITS4 primer.

As shown in Fig. 4 , the products detected in aseptic samples (Lane C) were also observed in Lane LNA(−) in all samples examined. Bands with the same mobility exhibited high intensity for wheat and soybean roots and the potato leaf and stem. This was presumably caused by the low fungal DNA/plant DNA ratio in the DNA extracted. Consequently, host plant DNAs were excessively amplified by the mismatch of the primer, even the ITS1F KU DNA primer, which had been designed specifically for fungi. However, when the ITS1F KU LNA primer was applied to these samples, the intensities of the host plant products decreased and the other bands with different sizes were additionally detected and/or their intensities increased (Lane 0 μM).

In contrast, the root and epidermises of young and mother potato tubers showed no bands of the same mobility as the aseptic samples when the ITS1F KU LNA primer was used in amplification, while other bands with different sizes were predominantly detected. This result suggests that fungal ITS regions are selectively amplified using the LNA primer only in the samples, which was considered to have a high fungal DNA/plant DNA ratio. Although faint bands giving similar locations to those of aseptic potato products were still detected in the epidermis of the young tuber (Lane 0 μM), they were imprecise. Fungal ITS regions are known to show different lengths, from approximately 400 bp to 900 bp ( 51 ); therefore, these bands were assumed to be fungal ITS products.

Thus, the application of an LNA primer enhanced the amplification of the ITS regions of plant–associated fungi more than a DNA primer. However, amplicons of host plant DNA were still detected at small intensities in samples that were considered to have a low fungal DNA/plant DNA ratio. In order to further inhibit the amplification of host plant DNA, the LNA oligonucleotide–PCR clamping technique was applied to compete with the ITS4 primer. LNA oligonucleotides a, b and c were used for wheat, soybean, and potato samples, respectively. As a result, the band intensities derived from host plants decreased with increases in the concentration of LNA oligonucleotides. No bands were observed at more than 2.0 μM, 3.0 μM, 6.0 μM, and 6.0 μM for the wheat root, soybean root, potato leaf, and potato stem, respectively, indicating that the combination of an LNA primer and the LNA oligonucleotide–PCR clamping technique effectively enhanced the amplification of fungal DNA, while inhibiting the amplification of host plant DNA.

Effects of the LNA technique in investigations of community structures of plant–associated fungi

The fungal PCR products that were selectively amplified using either an LNA primer or LNA primer and PCR clamping by LNA oligonucleotides were used for a fungal community analysis via DGGE in order to compare the patterns amplified by the conventional approach using ITS1F KU DNA and ITS4 primers. Nested PCR products were prepared from these products with the ITS1F KU DNA primer with the GC clamp (5′– CGCCCGCCGCGCGCGGCGGGCGGGGCGG GGGCACGGGGGG CTYGGTCATTTAGAGGAASTA A–3′, the underlined sequence indicates the GC clamp) and ITS2 primer (5′–GCTGCGTTCTTCATCGATGC–3′). The nested products were then used in the DGGE analysis to examine the effects of the LNA technique by comparing these patterns. In addition, DNA extracted from aseptic samples was directly amplified with the ITS1F KU DNA primer with the GC clamp and ITS2 primer in order to confirm the position of host plant amplicons in the DGGE gel. The ITS1F primer set with the GC clamp and ITS2 has frequently used been in community analyses of fungi in various environments ( 2 , 5 ), while the ITS1F sequence was modified to increase coverage in this study.

As shown in Fig. 5 , predominant DGGE bands giving the same mobility as aseptic samples were detected in wheat and soybean roots and in the potato leaf and stem amplicons prepared by the conventional approach without the LNA technique; however, several other bands derived from fungi were also observed in these patterns. This result indicated that the conventional approach underestimated the community structures of plant–associated fungi, particularly in samples with a low fungal DNA/plant DNA ratio.

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DGGE patterns of nested PCR products derived from respective parts of wheat, soybean, and potato samples. The products were prepared with the ITS1F KU DNA primer with the GC clamp and ITS2 primer. The symbols “−” and “+” indicate the lanes prepared without and with the LNA technique. The products that sufficiently amplified the fungal ITS regions are used in lane “+”. “C” represents aseptic amplicons to provide the position of the host plant band in the DGGE gel. The sequences of DGGE bands indicated with arrows were obtained in order to identify the closest relatives using the DNA database.

In contrast, when the LNA technique was applied, DGGE bands showing the same mobility as the host plants were not detected in the patterns of wheat and soybean roots, or the band intensities of host plants were significantly decreased in those of the potato leaf and stem. Furthermore, fungal DGGE bands were additionally detected at an acceptable level to investigate community structures. This was most likely due to fungal bands, which were hidden behind more strongly amplified host plant DNA, becoming visible on DGGE images.

In contrast to that described above, DGGE patterns generated from the roots and epidermises of young and mother potato tubers showed almost identical patterns between LNA(−) and LNA(+). These patterns only differed due to several bands showing greater intensities in the LNA(+) patterns than those in the LNA(−) patterns, and the band derived from host plant DNA was detected in the LNA(−) patterns. This result suggests the potential to investigate the community structures of plant–associated fungi without applying LNA primers when the host plant DNA band is faint. However, it is preferable to completely inhibit the amplification of host plant DNA by applying the LNA technique in order to obtain more accurate information.

Closest relatives of DGGE bands detected by applying the LNA technique

Some DGGE bands, newly detected using the LNA primer and PCR clamping by LNA oligonucleotides, were sequenced for wheat and soybean roots. As shown in Table 1 , all of the closest relatives belonged to fungi, and 15, 2, and 1 sequences were affiliated with Ascomycota , Basidiomycota , and unidentified fungi, respectively.

Closest relatives of DGGE bands that were detected using LNA technique

Of these, the closest relatives of bands W1, W2, W3, W4, S2, S4, S7, S8, S9, and S10 and W6, S1, and S12 were isolated from plants and agricultural soils, respectively. The closest relatives of bands W1, W3, W4, S8, and S11 are known to be fungi associated with wheat and soybean ( 4 , 23 , 28 , 40 , 52 , 54 ). Phoma sp. (closest relatives of bands W3 and S8) and Acremonium sp. (closest relatives of band S11) were reported to be beneficial for host plants through their roles in growth promotion and enhancements in resistance to insects and antimicrobial activity, respectively ( 7 , 38 , 58 ). The closest relatives of W2, W4, W6, S9, and S12 have not yet been isolated from wheat and soybean; however, Acidomelania panicicola (closest relative of band W2), Podospora glutinans (closest relative of band W4), and Scolecobasidium terreum (closest relatives of bands W6 and S12) were in the group of dark septate endophytic fungi ( 32 , 53 , 60 ) that contribute to plant growth in a number of ways ( 31 ).

Thus, the LNA technique–assisted community analysis is an effective method to obtain genetic information on plant– associated fungi, for which DNA had been screened by the conventional amplification of host plants DNA. This provides fundamental information for investigating the roles of fungi in plant growth, and subsequently to isolate fungi for further investigations, including the evaluation of microbial materials for agricultural applications.

The LNA oligonucleotides designed effectively amplify fungal DNA while suppressing the amplification of host plant DNA during PCR. However, LNA oligonucleotides may hybridize fungal DNA, the sequences of which are almost identical to host plant DNA. Consequently, the amplification of fungal DNA is inhibited, and the resultant DGGE patterns of plant–associated fungi may be affected. In order to investigate possible interference by LNA oligonucleotides in the amplification of fungal ITS regions, agricultural field soil, which contained an unspecified large variety of fungal DNA, was regarded as a sample, and DGGE patterns were compared between the amplicons prepared with and without LNA oligonucleotides (a, b, and c; added at 4.0 μM each). In the present study, PCR amplification was performed using ITS1F KU with the GC clamp, and ITS 2 after amplifying the whole ITS region with the ITS1F KU LNA primer, and ITS4 for extracted DNA.

As shown in Fig. 6 , the soil fungal DGGE patterns among lanes 1 to 4 were identical irrespective of the addition and types of LNA oligonucleotides. This result indicates that LNA oligonucleotides did not affect the amplification of fungal ITS regions, or, if they did, it was negligible for an investigation of fungal communities using a DGGE analysis.

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DGGE patterns of nested PCR products derived from agricultural soil. Products were prepared with the ITS1F KU DNA primer with the GC clamp and ITS2 primer after having amplified the whole ITS region with the ITS1F KU LNA primer and ITS4 primer for extracted DNA. LNA oligonucleotides a, b, and c were added to amplify the whole ITS region in order to examine possible interference in the amplification of fungal ITS regions during PCR. Lane 1 indicates the pattern prepared without LNA oligonucleotides, while lanes 2, 3, and 4 indicate those prepared with LNA oligonucleotides a, b, and c, respectively.

LNA primers and PCR clamping by LNA oligonucleotides were applied to enhance the amplification of fungal ITS regions for investigations of plant–associated community structures using DNA extracted from plant samples containing eukaryotic DNA, particularly host plant DNA. Overall, the application of LNA primers enhanced the amplification of fungal DNA. LNA primers sufficiently amplified the fungal ITS region in samples, presumably with a high fungal DNA/plant DNA ratio. On the other hand, the combination of LNA primers and PCR clamping by LNA oligonucleotides was necessary for samples with a low fungal DNA/plant DNA ratio. A DGGE analysis showed that fungal banding patterns reached an acceptable level for investigating the community structures of plant–associated fungi. The closest relatives of the DGGE bands all belonged to fungi and some were assumed to contribute to plant growth in a number of ways. Thus, the LNA technique assisted in a fungal community analysis of plant–associated fungi. This amplification technique will benefit further investigations.

Supplementary Information

Acknowledgements.

We would like to thank Dr. Isao AKAGI of Kagoshima University for his advice on plant cultivation, and Dr. Tsuyoshi TOMIHAMA of the Kagoshima Prefectural Institute for Agricultural Development for providing potato tubers in this ongoing study. This work was mostly supported by the Council for Science, Technology and Innovation (CSTI), Cross–ministerial Strategic Innovation Promotion Program (SIP), and “Technologies for creating next generation agriculture, forestry and fisheries” (funding agency: Bio–oriented Technology Research Advancement Institution, NARO).

How to Write a Term Paper in 5 Steps

Matt Ellis

Term papers are a key way to test a student’s knowledge and research skills, but they can be difficult to write. In this guide, we explain the best methods to write a term paper, including the proper term paper format and even how to choose a term paper topic.

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What is a term paper?

A term paper is a piece of academic writing in which a student demonstrates their knowledge of a topic of study. Term papers constitute a large portion of the final grade, making them a serious assignment. There is typically no more than one term paper assigned each term, although how long a term lasts depends on the school system.

Keep in mind that a term paper is one specific type of academic paper. It is more intensive than a standard writing assignment but is not as in-depth as a thesis paper or dissertation.

How long is a term paper?

There is no standard length for a term paper; each subject, course, and professor has their own preferences. Term papers can be as short as five pages or as long as twenty pages, but they usually fall somewhere in the middle.

What’s the difference between a term paper and a research paper?

Technically speaking, a research paper is a paper that argues its main point with original data and evidence. However, the term research paper is used informally to refer to any paper that requires research, even when collecting data and evidence from other preexisting sources. So in that sense, a term paper can be a research paper if the student must research other sources to complete it.

The terms term paper and research paper are often used interchangeably. However, term papers are generally assigned once per term, whereas a teacher or professor can assign as many research papers as they wish.

What’s the difference between a term paper and an essay?

An essay is any writing that asserts the author’s opinion or perspective, whether for school, publication, or just the author’s personal enjoyment. Unlike research-oriented term papers that draw from data and evidence, essay writing is based only on the author’s experience or viewpoint.

Essays are usually shorter than term papers and more casual in tone. Keep in mind that term papers are strictly academic, whereas essays can be written for various audiences.

How do I write a term paper?

Writing a term paper still follows the standard writing process but with some extra focus in certain areas.

1 Developing ideas

The first step of writing a term paper is brainstorming to come up with potential topics and then selecting the best one. Sometimes your topics are assigned, but often you’ll have to choose one yourself.

In addition to picking a topic that you’re personally interested in, try to settle on one that has sufficient depth. Avoid topics that are too broad because you won’t be able to cover everything, and stay away from topics that are too specific because you may not find enough information to fill the required paper length.

If you’re looking for inspiration, check out our list of term and research paper topics .

2 Preparation (research)

The preparation stage is when you determine your main point and the parts of your topic you’re going to discuss. For most term papers, that requires research. If you’re not conducting your own research, then you’re finding and reviewing sources to use instead.

A good place to start is by writing your thesis statement , a single sentence that sums up the main point(s) your paper tries to make. Your thesis statement determines what evidence and counterarguments you’ll need to discuss. Deciding on these early can help streamline your research.

Once you establish what you want to include in your term paper, you can start putting it in order by writing an outline . Think of the outline as the blueprint of your term paper, mapping out each part of your topic, paragraph by paragraph.

Be sure to follow the term paper format for the assignment. This means adhering to the guidelines and planning enough content to meet the length requirement.

4 First draft

Writing the first draft is easier if you follow your outline. Although this stage can be the most labor-intensive, remember that everything doesn’t need to be perfect. You can still go back later to revise and optimize your wording, but for the first draft, just focus on getting all your ideas down on paper.

This isn’t always easy. If you’re having trouble or get stuck at certain points, go back to the fundamentals and revisit your first-year writing skills. If you have writer’s block, don’t be afraid to take a break and try again later—your brain could just be too tired to come up with ideas.

5 Editing and proofreading

After you have completed a first draft, it’s time to begin the editing process. This is when you correct the mistakes in the first draft and detect other issues that need revising. If a section seems weak or inadequate, you can revise the wording or even rewrite it entirely. You may find that something is missing from your first draft, so now is the time to add it.

We recommend rereading your term paper twice—once to correct the wording and structural mistakes and another time to proofread . Revising it twice allows you to better focus on particular issues instead of trying to address everything at once. If you’re trying to determine the right word choice , spending time on spelling and grammar might be a distraction. It’s better to separate the tasks and do them one at a time.

Term paper FAQs

How do i write my term paper.

Writing a term paper still follows the standard writing process, but goes deeper into certain areas. Start by brainstorming topics that you find interesting before selecting one that has ample source material. Then begin your research. When you’re ready to start writing, create an outline, then a first draft, and finally revisions.

There is no standard length for a term paper; every teacher or professor has their own requirements. Term papers can be as short as five pages or as long as twenty pages, but they usually fall somewhere in the middle.

Technically speaking, a research paper supports its thesis with original data and evidence. However, the term research paper is used informally to refer to any paper that requires research, even when collecting data and evidence from other preexisting sources. So in that sense, a term paper can also be a research paper if the student relies on other sources to complete it.

term paper on lna

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HHS Statement Regarding the Cyberattack on Change Healthcare

The U.S. Department of Health and Human Services (HHS) is aware that Change Healthcare – a unit of UnitedHealth Group (UHG) – was impacted by a cybersecurity incident in late February. HHS recognizes the impact this attack has had on health care operations across the country. HHS’ first priority is to help coordinate efforts to avoid disruptions to care throughout the health care system.

HHS is in regular contact with UHG leadership, state partners, and with numerous external stakeholders to better understand the nature of the impacts and to ensure the effectiveness of UHG’s response. HHS has made clear its expectation that UHG does everything in its power to ensure continuity of operations for all health care providers impacted and HHS appreciates UHG’s continuous efforts to do so. HHS is also leading interagency coordination of the Federal government’s related activities, including working closely with the Federal Bureau of Investigations (FBI), the Cybersecurity and Infrastructure Security Agency (CISA), the White House, and other agencies to provide credible, actionable threat intelligence to industry wherever possible.

HHS refers directly to UHG for updates on their incident response progress and recovery planning. However, numerous hospitals, doctors, pharmacies and other stakeholders have highlighted potential cash flow concerns to HHS stemming from an inability to submit claims and receive payments. HHS has heard these concerns and is taking direct action and working to support the important needs of the health care community.

Today, HHS is announcing immediate steps that the Centers for Medicare & Medicaid Services (CMS) is taking to assist providers to continue to serve patients. CMS will continue to communicate with the health care community and assist, as appropriate. Providers should continue to work with all their payers for the latest updates on how to receive timely payments.

Affected parties should be aware of the following flexibilities in place:

  • Medicare providers needing to change clearinghouses that they use for claims processing during these outages should contact their Medicare Administrative Contractor (MAC) to request a new electronic data interchange (EDI) enrollment for the switch. The MAC will provide instructions based on the specific request to expedite the new EDI enrollment. CMS has instructed the MACs to expedite this process and move all provider and facility requests into production and ready to bill claims quickly. CMS is strongly encouraging other payers, including state Medicaid and Children’s Health Insurance Program (CHIP) agencies and Medicaid and CHIP managed care plans, to waive or expedite solutions for this requirement.
  • CMS will issue guidance to Medicare Advantage (MA) organizations and Part D sponsors encouraging them to remove or relax prior authorization, other utilization management, and timely filing requirements during these system outages. CMS is also encouraging MA plans to offer advance funding to providers most affected by this cyberattack.
  • CMS strongly encourages Medicaid and CHIP managed care plans to adopt the same strategies of removing or relaxing prior authorization and utilization management requirements, and consider offering advance funding to providers, on behalf of Medicaid and CHIP managed care enrollees to the extent permitted by the State. 
  • If Medicare providers are having trouble filing claims or other necessary notices or other submissions, they should contact their MAC for details on exceptions, waivers, or extensions, or contact CMS regarding quality reporting programs.
  • CMS has contacted all of the MACs to make sure they are prepared to accept paper claims from providers who need to file them. While we recognize that electronic billing is preferable for everyone, the MACs must accept paper submissions if a provider needs to file claims in that method.

CMS has also heard from providers about the availability of accelerated payments, like those issued during the COVID-19 pandemic. We understand that many payers are making funds available while billing systems are offline, and providers should take advantage of those opportunities. However, CMS recognizes that hospitals may face significant cash flow problems from the unusual circumstances impacting hospitals’ operations, and – during outages arising from this event – facilities may submit accelerated payment requests to their respective servicing MACs for individual consideration. We are working to provide additional information to the MACs about the specific items and information a provider’s request should contain. Specific information will be available from the MACs later this week.

This incident is a reminder of the interconnectedness of the domestic health care ecosystem and of the urgency of strengthening cybersecurity resiliency across the ecosystem. That’s why, in December 2023, HHS released a concept paper that outlines the Department’s cybersecurity strategy for the sector. The concept paper builds on the National Cybersecurity Strategy that President Biden released last year, focusing specifically on strengthening resilience for hospitals, patients, and communities threatened by cyber-attacks. The paper details four pillars for action, including publishing new voluntary health care-specific cybersecurity performance goals, working with Congress to develop supports and incentives for domestic hospitals to improve cybersecurity, increasing accountability within the health care sector, and enhancing coordination through a one-stop shop.

HHS will continue to communicate with the health care sector and encourage continued dialogue among affected parties. We will continue to communicate with UHG, closely monitor their ongoing response to this cyberattack, and promote transparent, robust response while working with the industry to close any gaps that remain.

HHS also takes this opportunity to encourage all providers, technology vendors, and members of the health care ecosystem to double down on cybersecurity, with urgency. The system and the American people can ill afford further disruptions in care. Please visit the  HPH Cyber Performance Goals website for more details on steps to stay protected.

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Reflecting on Cybersecurity Awareness Month

Navigating section 752: insights from program managers on success, challenges, and tools for change, thank you to the 2023 civic digital fellows, media inquiries.

For general media inquiries, please contact  [email protected] .

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Russia’s 2024 Presidential Vote: What to Know

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By Neil MacFarquhar

Why does this vote matter?

Does putin face any serious challengers, will the kremlin manipulate the results, can russians protest, can putin remain president for life.

When will the results be known?

Where can I find more information?

The presidential vote in Russia, which began Friday and lasts through Sunday, features the trappings of a horse race but is more of a predetermined, Soviet-style referendum.

President Vladimir V. Putin, 71, will undoubtedly win a fifth term, with none of the three other candidates who are permitted on the ballot presenting a real challenge. The main opposition figure who worked to spoil the vote, Aleksei A. Navalny , a harsh critic of Mr. Putin and the Ukraine war, died in an Arctic prison last month.

Still, the vote is significant for Mr. Putin as a way to cement his legitimacy and refurbish his preferred image as the embodiment of security and stability. That image was tarnished when the war, advertised as a speedy operation to topple the government in Kyiv, turned into a slog that caused hundreds of thousands of casualties, ruptured relations with the West and ushered in harsher domestic repression.

“The Kremlin needs to demonstrate huge popular support, and that this support has increased since the beginning of the war,” said Nikolay Petrov, a Russian political scientist at the German Institute for International and Security Affairs in Berlin.

The Kremlin habitually ensures that Mr. Putin faces no real competition. The other candidates — all members of the State Duma, Russia’s rubber-stamp Parliament — voted for the war in Ukraine, for increased censorship and for laws curbing gay rights.

Nikolai Kharitonov, 75, of the Communist Party, already lost badly to Mr. Putin in 2004.

Leonid Slutsky, 56 of the Liberal Democratic Party, a nationalist group loyal to Mr. Putin, has said he will not rally voters against the president.

Vladislav A. Davankov, 40, from the New People Party, is nominally liberal and has called for “peace” in Ukraine but has basically supported Mr. Putin.

Two candidates opposed to the war were disqualified. A veteran politician, Boris Nadezhdin , alarmed the Putin administration when tens of thousands of people across Russia lined up to sign petitions required for him to run. The Kremlin invalidated enough signatures to bar him.

Russia held real elections for about a decade after the Soviet Union collapsed in 1991. Ever since, the Kremlin has relied on various social, geographic and technical levers to ensure that its candidate receives an overwhelming majority.

Although Mr. Putin enjoys some support, the Kremlin has long sought to proclaim that he received more than 50 percent support in balloting, and also more support than he did in each previous vote. This year that means outstripping the 56 million votes that the authorities said he received in 2018; pundits are betting on 60 million.

Two important changes this time could add to the vote’s opacity.

For one, balloting will be held in the so-called “new territories,” the four Ukrainian regions Moscow annexed without fully controlling them. Russia’s election officials say the area has 4.5 million voters, an assertion virtually impossible to monitor amid a war.

“We cannot check the figures there and the authorities will use them as they wish,” said Alexander V. Kynev, an independent election expert in Moscow.

Also, the ability to vote online will be more widely available, with electronic voters in 29 regions on one huge list, with no means to check where or how they voted, Mr. Kynev noted.

In a sprawling, diverse country like Russia, the Kremlin can also use more traditional means. Regions dominated by ethnic strongmen, like the Caucasus, habitually report huge turnouts with Mr. Putin receiving 99 percent of the vote — even if relatively few people show up at polling stations.

Areas where state industries prevail also tend to report heavy support for the president. To turn out the vote, some polling stations hold raffles for prizes like household appliances or firewood. One Siberian region is offering 16,000 prizes.

But the Kremlin must rely on some votes in big cities, and that can get tricky. Excessive manipulation has created unrest previously. There might be slightly more manipulation this year because monitors are barred unless issued credentials by the candidates.

With street demonstrations banned, some Putin opponents hope to cast protest votes. The simplest method to lower his tally is to vote for someone else, experts noted.

“Noon Against Putin,” a campaign pushed by Mr. Navalny’s organization, suggests swarming polling places at midday on Sunday. But there are a number of hurdles, including possible confrontations with the police.

Also, in previous votes, few polling stations had more than 3,000 registered voters and many had fewer than 1,000. “It is technically very complicated to create a crowd,” said David Kankiia, an analyst with the Golos election watchdog, barred in Russia.

Since he was first appointed successor to President Boris Yeltsin in 2000, Mr. Putin has said Russia’s Constitution would dictate the length of his tenure. Then he kept rewriting the Constitution.

Asked in 2014 whether he would remain president forever, Mr. Putin responded , “This is not good and it is detrimental for the country and I do not need it either,” before adding, “We will see what the situation will be like, but in any case the term of my work is restricted by the Constitution.”

In 2008, when term limits forced him to step aside, he became prime minister under President Dmitri A. Medvedev, although Mr. Putin remained the power behind the throne until reclaiming the top job in 2012.

Presidential terms were extended to six years before the 2018 vote, and then in 2020 Mr. Putin changed the constitution again to reset his term clock. At this point, he can have at least two terms until 2036. If Mr. Putin lasts, he will soon outstrip the record, 29-year rule of Joseph Stalin.

When will the voting results be known?

The tally is expected to be announced sometime Sunday night Moscow time.

Putin, in Pre-Election Messaging, Is Less Strident on Nuclear War

A Collective ‘No’: Anti-Putin Russians Embrace an Unlikely Challenger

Russia Bars Antiwar Candidate in Election Putin Is All But Sure of Winning

Milana Mazaeva , Alina Lobzina and Oleg Matsnev contributed reporting.

An earlier version of this article misstated the year in which Vladimir V. Putin returned to the presidency. It was 2012, not 2014.

How we handle corrections

Neil MacFarquhar has been a Times reporter since 1995, writing about a range of topics from war to politics to the arts, both internationally and in the United States. More about Neil MacFarquhar

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  1. Review article Locked nucleic acid (LNA): A modern approach to cancer diagnosis and treatment

    As the basic furanose flexibility in LNA has been locked, this bicyclic structure is known as locked nucleic acid [3].More precisely, due to the bridge that connects 2′-oxygen to 4′-carbon in the sugar structure, this group of nucleic acid analog is called locked nucleic acid [5], leading to a locked ribose group in the C3′-endo (North type) conformation (Fig. 1 a).

  2. Locked nucleic acid (LNA): High affinity targeting of RNA for

    Locked nucleic acid (LNA) comprises a class of RNA analogues in which the furanose ring of the ribose sugar is chemically locked in an RNA-mimicking conformation by the introduction of a O 2′, C 4′-methylene linkage [ 1, 2 ]. Several studies have demonstrated that LNA-modified oligonucleotides exhibit unprecedented thermal stabilities when ...

  3. LNA blockers for improved amplification selectivity

    Abstract. LNA-containing oligonucleotides bind DNA more tightly than standard DNA, so they can interact with targeted sequences and affect multiple processes. When a desired DNA is present at low ...

  4. Position-dependent effects of locked nucleic acid (LNA) on DNA

    LNA is a bicyclic ribose derivative with a bridging methylene group between O-2′ and C-4′. LNA provides the largest known increase in thermal stability of any modified DNA duplex , because it reduces the unfavorable entropy of duplex formation and may improve base stacking. LNA also has greater mismatch sensitivity than DNA (5, 6). LNA is ...

  5. LNA blockers for improved amplification selectivity

    Introduction. Locked Nucleic Acid (LNA) nucleotides are identical to natural nucleotides except for a methylene bridge spanning the deoxyribose sugar 1, 2 which makes them more stable in double-stranded structures and more resistant to degradation 3.The higher melting temperatures (T m s) of oligonucleotides that include LNA bases provide greater specificity and new functions.

  6. Electronics

    The challenging task of the LNA design is to provide equitable trade-off performances such as gain, power consumption, the noise figure, stability, linearity, and impedance matching. The design of fast settling LNA for a duty-cycled WuRx front-end operating at a 868 MHz frequency band is investigated in this work. The paper details the trade ...

  7. Design and performance analysis of low power LNA with ...

    CG LNA with input T matching circuit is shown in Fig. 1(a), which is designed for wideband and resonance of inductors \({L}_{1}\) and \({L}_{2}\) with parasitic capacitances gives flat gain. Designed LNA has less power consumption of 1mW but having large area 0.73 mm 2 and low gain of 7.9 dB so not usable for area sensitive applications.

  8. Locked Nucleic Acid

    LNA oligonucleotides (ONs) contain one or more LNA ON monomer(s) (Figure 2.1), which is a ribonucleotide analogue where the 2′-oxygen and the 4′-carbon atoms are connected via a methylene bridge (Obika et al., 1997; Singh, Nielsen, et al., 1998).This bridge locks the sugar moiety in an N-type sugar ring conformation such that LNA can be considered an RNA mimic.

  9. Amplification and Re-Generation of LNA-Modified Libraries

    Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide ...

  10. Locked nucleic acid (LNA) mediated improvements in siRNA stability and

    The LNA substitutions at position 10 and 14 exchanged an RNA-U for an LNA-T and an RNA-C for an LNA-m C both of which lead to the introduction of an additional methyl-group on the nucleobase. In the A-form helix formed between the siRNA/RNA-target, these methyl groups will protrude into the major groove with potential effects on helical ...

  11. Comparative Analysis Of LNA Based On Different Topologies And

    LNA is an electronic amplifier that amplifies very low power signal with significantly not degrading the SNR ratio. The basic requirements of LNA are to minimize the noise coming from the device, provide high Linearity, very low power consumption, high Gain and matching. ... Students looking for free, top-notch essay and term paper samples on ...

  12. What is LNA and Why is it Such a Powerful Research Tool?

    What are locked nucleic acids (LNA)? Locked nucleic acids are a class of high-affinity RNA analogs in which the ribose ring is "locked" in the ideal conformation for Watson-Crick binding (see figure Structure of LNA). Structure of LNA. The ribose ring is connected by a methylene bridge between the 2'-O and 4'-C atoms, "locking" the ribose ...

  13. An astute synthesis of locked nucleic acid monomers

    Seminal papers on LNA were independently instigated by Wengel [9,10] and Imanishi [] groups.It is well known that the B-form DNA duplex possesses C 2′-endo (S-type) and the A-form RNA duplex has C 3′-endo (N-type) sugar puckering [12,13].LNA is considered to be RNA mimic as the ancillary methylene bridge locks the sugar moiety into N-type sugar ring conformation (Figure 1).

  14. Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA

    2.. LNAIn 1998, the Wengel [10], [11] and Imanishi [12] laboratories described oligomer synthesis and hybridization using a novel nucleotide termed LNA (Fig. 1), with a subsequent report from the Wang laboratory appearing in 1999 [13].LNA nucleotides contain a methylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon. This bridge results in a locked 3′-endo conformation ...

  15. Locked nucleic acid

    Chemical structure of an LNA monomer an additional bridge bonds the 2' oxygen and the 4' carbon of the pentose. A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. . The bridge "locks" the ribose in the 3 ...

  16. LNA: a versatile tool for therapeutics and genomics

    Locked nucleic acid (LNA) is a nucleic acid analogue that displays unprecedented hybridization affinity towards complementary DNA and RNA. Structural studies have shown LNA to be an RNA mimic, fitting seamlessly into an A-type duplex geometry. Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide ...

  17. Term Paper

    Term Paper. Definition: Term paper is a type of academic writing assignment that is typically assigned to students at the end of a semester or term. It is usually a research-based paper that is meant to demonstrate the student's understanding of a particular topic, as well as their ability to analyze and synthesize information from various sources.. Term papers are usually longer than other ...

  18. Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids

    1. INTRODUCTION. The themodynamics 1-7 and kinetics 8-12 of Watson—Crick hybridization and strand displacement are well known for DNA and RNA oligonucleotides. As an alternative to naturally occurring nucleic acids, locked nucleic acids (LNAs) are conformationally restricted RNA nucleotides, where the 2′ oxygen in the ribose bonds to the 4′ carbon. 13-16 This covalent bond ...

  19. How to Write a Term Paper: Step-by-Step Guide With Examples

    4. Write your abstract. Because the abstract is a summary of your entire paper, it's usually best to write it after you complete your first draft. Typically, an abstract is only 150-250 words, so focus on highlighting the key elements of your term paper like your thesis, main supporting evidence, and findings.

  20. How To Become An LNA

    2.Pass your exam. Passing the LNA or CNA exam is usually required to practice as an LNA. Depending on your state, the test will be either a multiple-choice test or a multi-faceted test with written requirements. Once you've passed the exam, you'll receive your certification and can being practicing as an LNA.

  21. How to Write a Term Paper From Start to Finish

    A term paper is typically given at the conclusion of a course, serving as a comprehensive summary of the knowledge acquired during that term. It follows a structured format and may delve into specific topics covered within the course. On the other hand, a research paper delves deeper, involving original research, thorough analysis, and the ...

  22. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA

    LNA oligonucleotides a, b, and c were added to amplify the whole ITS region in order to examine possible interference in the amplification of fungal ITS regions during PCR. Lane 1 indicates the pattern prepared without LNA oligonucleotides, while lanes 2, 3, and 4 indicate those prepared with LNA oligonucleotides a, b, and c, respectively. ...

  23. How to Write a Term Paper in 5 Steps

    1 Developing ideas. The first step of writing a term paper is brainstorming to come up with potential topics and then selecting the best one. Sometimes your topics are assigned, but often you'll have to choose one yourself. In addition to picking a topic that you're personally interested in, try to settle on one that has sufficient depth.

  24. HHS Statement Regarding the Cyberattack on Change Healthcare

    FOR IMMEDIATE RELEASE March 5, 2024. Contact: HHS Press Office 202-690-6343 [email protected]. HHS Statement Regarding the Cyberattack on Change Healthcare. The U.S. Department of Health and Human Services (HHS) is aware that Change Healthcare - a unit of UnitedHealth Group (UHG) - was impacted by a cybersecurity incident in late February.

  25. Russia's 2024 Presidential Vote: What to Know

    President Vladimir V. Putin, 71, will undoubtedly win a fifth term, with none of the three other candidates who are permitted on the ballot presenting a real challenge. The main opposition figure ...