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PCR-based techniques are methods that rely on the polymerase chain reaction (PCR) to amplify stretches of DNA by creating many identical or near-identical copies. For example, PCR amplification can be used to isolate the sequence at a specific location in the genome (genotyping).

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• Published: 19 January 2015

Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering

• Sayed Shahabuddin Hoseini 1 , 2 &
• Martin G Sauer 1 , 2 , 3  

Journal of Biological Engineering volume  9 , Article number:  2 ( 2015 ) Cite this article

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Over the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning.

Exemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells.

Conclusions

Using a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.

Various techniques were introduced for assembling new DNA sequences [ 1 – 3 ], yet the use of restriction endonuclease enzymes is the most widely used technique in molecular cloning. Whenever compatible restriction enzyme sites are available on both, insert and vector DNA sequences, cloning is straightforward; however, if restriction sites are incompatible or if there is even no restriction site available in the vicinity of the insert cassette, cloning might become more complex. The use of PCR primers, in which compatible restriction enzyme sites are embedded, can effectively solve this problem and facilitate multistep cloning procedures.

Although PCR cloning has been vastly used in biological engineering [ 4 – 8 ], practical guides explaining all necessary steps and tips in a consecutive order are scarce. Furthermore, the emergence of new high-fidelity DNA polymerases, kits, and powerful software makes the process of PCR cloning extremely fast and efficient. Here we sequentially explain PCR cloning from the analysis of the respective gene sequence, the design of PCR primers, performing the PCR procedure itself, sequencing the resulting PCR products, analysis of sequencing data, and finally the cloning of the PCR product into the final vector.

Results and discussion

Choosing proper restriction enzymes based on defined criteria.

In order to proceed with a concise example, tdTomato fluorescent protein was cloned into an alpharetroviral vector. Consecutively, a murine leukemia cell line expressing tdTomato was generated. This cell line will be used to track tumor cells upon injection into mice in preclinical immunotherapy studies. However, this cloning method is applicable to any other gene. To begin the cloning project, the gene of interest (GOI) should be analyzed. First, we check whether our annotated sequence has a start codon (ATG, the most common start codon) and one of the three stop codons (TAA, TAG, TGA). In case the gene was previously manipulated or fused to another gene (e.g. via a 2A sequence), it happens that a gene of interest might not have a stop codon [ 9 ]. In such cases, a stop codon needs to be added to the end of your annotated sequence. It is also beneficial to investigate whether your GOI contains an open reading frame (ORF). This is important since frequent manipulation of sequences either by software or via cloning might erroneously add or delete nucleotides. We use Clone Manager software (SciEd) to find ORFs in our plasmid sequences; however, there are several free websites you can use to find ORFs including the NCBI open reading frame finder ( http://www.ncbi.nlm.nih.gov/gorf/gorf.html ).

The tdTomato gene contains ATG start codon and TAA stop codon (Figure  1 ). The size of the tdTomato gene is 716 bp.

Overview of the start and the end of the gene of interest. (A) The nucleotide sequences at the start and the end of the tdTomato gene are shown. The coding strand nucleotides are specified in bold (B) The nucleotide sequences of the forward and reverse primers containing proper restriction enzyme sites and the Kozak sequence are shown.

In a next step, PCR primers that include proper restriction enzyme sites need to be designed for the amplification of the GOI. Several criteria should be considered in order to choose the optimal restriction enzymes. First, binding sites for restriction enzymes should be ideally available at a multiple cloning site within the vector. Alternatively they can be located downstream of the promoter in your vector sequence. Restriction enzymes should be single cutters (single cutters target one restriction site only within a DNA sequence) (Figure  2 A). If they are double or multiple cutters, they should cut within a sequence that is not necessary for proper functioning of the vector plasmid and will finally be removed (Figure  2 B). It is also possible to choose one double cutter or multiple cutter enzymes cutting the vector downstream of the promoter and also not within a vital sequence of the plasmid (Figure  2 C). Double cutter or multiple cutter enzymes have two or more restriction sites on a DNA sequence, respectively. Cutting the vector with double or multiple cutters would give rise to two identical ends. In such a case, the insert cassette should also contain the same restriction enzyme sites on both of its ends. Therefore, when the insert and vector fragments are mixed in a ligation experiment, the insert can fuse to the vector in either the right orientation (from start codon to stop codon) or reversely (from stop codon to start codon). A third scenario can occur, if the vector fragment forms a self-ligating circle omitting the insert at all. Once the DNA has been incubated with restriction enzymes, dephosphorylation of the 5′ and 3′ ends of the vector plasmid using an alkaline phosphatase enzyme will greatly reduce the risk of self-ligation [ 10 ]. It is therefore important to screen a cloning product for those three products (right orientation, reverse orientation, self-ligation) after fragment ligation.

Choosing proper restriction enzymes based on defined criteria for PCR cloning. (A) Two single-cutter restriction enzymes (E1 and E2) are located downstream of the promoter. (B) E1 and E2 restriction enzymes cut the plasmid downstream of the promoter several (here two times for each enzyme) times. (C) The E1 restriction enzyme cuts the plasmid downstream of the promoter more than once. (D) The PCR product, which contains the tdTomato gene and the restriction enzyme sites, was run on a gel before being extracted for downstream applications.

Second, due to higher cloning efficiency using sticky-end DNA fragments, it is desirable that at least one (better both) of the restriction enzymes is a so-called sticky-end cutter. Sticky end cutters cleave DNA asymmetrically generating complementary cohesive ends. In contrast, blunt end cutters cut the sequence symmetrically leaving no overhangs. Cloning blunt-end fragments is more difficult. Nevertheless, choosing a higher insert/vector molar ratio (5 or more) and the use 10% polyethylene glycol (PEG) can improve ligation of blunt-end fragments [ 11 ].

Third, some restriction enzymes do not cut methylated DNA. Most of the strains of E. coli contain Dam or Dcm methylases that methylate DNA sequences. This makes them resistant to methylation-sensitive restriction enzymes [ 12 ]. Since vector DNA is mostly prepared in E. coli , it will be methylated. Therefore avoiding methylation-sensitive restriction enzymes is desirable; however, sometimes the isoschizomer of a methylation-sensitive restriction enzyme is resistant to methylation. For example, the Acc 65I enzyme is sensitive while its isoschizomer kpn I is resistant to methylation [ 13 ]. Isoschizomers are restriction enzymes that recognize the same nucleotide sequences. If there remains no other option than using methylation-sensitive restriction enzymes, the vector DNA needs to be prepared in dam − dcm − E. coli strains. A list of these strains and also common E. coli host strains for molecular cloning is summarized in Table  1 . Information regarding the methylation sensitivity of restriction enzymes is usually provided by the manufacturer.

Fourth, it makes cloning easier if the buffer necessary for the full functionality of restriction enzymes is the same because one can perform double restriction digest. This saves time and reduces the DNA loss during purification. It may happen that one of the restriction enzymes is active in one buffer and the second enzyme is active in twice the concentration of the same buffer. For example the Nhe I enzyme from Thermo Scientific is active in Tango 1X buffer (Thermo Scientific) and Eco R1 enzyme is active in Tango 2X buffer (Thermo Scientific). In such cases, the plasmid DNA needs to be first digested by the enzyme requiring the higher buffer concentration (here Eco R1). This will be followed by diluting the buffer for the next enzyme (requiring a lower concentration (here Nhe I)) in the same buffer. However, the emergence of universal buffers has simplified the double digest of DNA sequences [ 15 ]. In our example the vector contains the Age I and Sal I restriction sites. These enzyme sites were used for designing PCR primers (Figure  1 ). It is essential for proper restriction enzyme digestion that the plasmid purity is high. DNA absorbance as measured by a spectrophotometer can be used to determine the purity after purification. DNA, proteins, and solvents absorb at 260 nm, 280 nm, and 230 nm, respectively. An OD 260/280 ratio of >1.8 and an OD 260/230 ratio of 2 to 2.2 is considered to be pure for DNA samples [ 16 ]. The OD 260/280 and 260/230 ratios of our exemplary plasmid preparations were 1.89 and 2.22, respectively. We observed that the purity of the gel-extracted vector and insert DNA fragments were lower after restriction digest; ligation works even in such cases, however, better results can be expected using high-purity fragments.

The following plasmid repository website can be useful for the selection of different vectors (viral expression and packaging, empty backbones, fluorescent proteins, inducible vectors, epitope tags, fusion proteins, reporter genes, species-specific expression systems, selection markers, promoters, shRNA expression and genome engineering): http://www.addgene.org/browse/ .

A collection of cloning vectors of E. coli is available under the following website: http://www.shigen.nig.ac.jp/ecoli/strain/cvector/cvectorExplanation.jsp .

Designing cloning primers based on defined criteria

For PCR primer design, check the start and stop codons of your GOI. Find the sequence of the desired restriction enzymes (available on the manufacturers’ websites) for the forward primer (Figure  3 A). It needs to be located before the GOI (Figure  1 B). The so-called Kozak sequence is found in eukaryotic mRNAs and improves the initiation of translation [ 17 ]. It is beneficial to add the Kozak sequence (GCCACC) before the ATG start codon since it increases translation and expression of the protein of interest in eukaryotes [ 18 ]. Therefore, we inserted GCCACC immediately after the restriction enzyme sequence Age I and before the ATG start codon. Then, the first 18 to 30 nucleotides of the GOI starting from the ATG start codon are added to the forward primer sequence. These overlapping nucleotides binding to the template DNA determine the annealing temperature (Tm). The latter is usually higher than 60°C. Here, we use Phusion high-fidelity DNA polymerase (Thermo Scientific). You can use the following websites for determination of the optimal Tm: http://www.thermoscientificbio.com/webtools/tmc/ .

Designing primers based on defined criteria for PCR cloning. (A-B) Sequences of the forward and the reverse primer are depicted. The end of the coding strand is to be converted into the reverse complement format for the reverse primer design. For more information, please see the text.

https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator .

The Tm of our forward primer is 66°C.

Choose the last 18 to 30 nucleotides including the stop codon of your GOI for designing the reverse primer (Figure  3 B). Then calculate the Tm for this sequence which should be above 60°C and close to the Tm of the forward primer. Tm of the overlapping sequence of our reverse primer was 68°C. Then, add the target sequence of the second restriction enzyme site (in this case Sal I) immediately after the stop codon. Finally, convert this assembled sequence to a reverse-complement sequence. The following websites can be used to determine the sequence of the reverse primer:

http://reverse-complement.com/

http://www.bioinformatics.org/sms/rev_comp.html This is important since the reverse primer binds the coding strand and therefore its sequence (5′ → 3′) must be reverse-complementary to the sequence of the coding strand (Figure  1 A).

Performing PCR using proofreading polymerases

Since the PCR reaction follows logarithmic amplification of the target sequence, any replication error during this process will be amplified. The error rate of non-proofreading DNA polymerases, such as the Taq polymerase, is about 8 × 10 −6 errors/bp/PCR cycle [ 19 ]; however, proofreading enzymes such as Phusion polymerase have a reported error rate of 4.4 × 10 −7 errors/bp/PCR cycle. Due to its superior fidelity and processivity [ 20 – 22 ], the Phusion DNA polymerase was used in this example. It should be noted that Phusion has different temperature requirements than other DNA polymerases. The primer Tm for Phusion is calculated based on the Breslauer method [ 23 ] and is higher than the Tm using Taq or pfu polymerases. To have optimal results, the Tm should be calculated based on information found on the website of the enzyme providers. Furthermore, due to the higher speed of Phusion, 15 to 30 seconds are usually enough for the amplification of each kb of the sequence of interest.

After the PCR, the product needs to be loaded on a gel (Figure  2 D). The corresponding band needs to be cut and the DNA extracted. It is essential to sequence the PCR product since the PCR product might include mutations. There are several PCR cloning kits available some of which are shown in Table  2 . We used the pJET1.2/blunt cloning vector (Thermo Scientific, patent publication: US 2009/0042249 A1, Genbank accession number EF694056.1) and cloned the PCR product into the linearized vector. This vector contains a lethal gene ( eco47IR ) that is activated in case the vector becomes circularized. However, if the PCR product is cloned into the cloning site within the lethal gene, the latter is disrupted allowing bacteria to grow colonies upon transformation. Circularized vectors not containing the PCR product express the toxic gene, which therefore kills bacteria precluding the formation of colonies. Bacterial clones are then to be cultured, plasmid DNA consecutively isolated and sequenced. The quality of isolated plasmid is essential for optimal sequencing results. We isolated the plasmid DNA from a total of 1.5 ml cultured bacteria (yield 6 μg DNA; OD 260/280 = 1.86; OD 260/230 = 2.17) using a plasmid mini-preparation kit (QIAGEN). The whole process of PCR, including cloning of the PCR product into the sequencing vector and transfection of bacteria with the sequencing vector can be done in one day. The next day, bacterial clones will be cultured overnight before being sent for sequencing.

Analysis of sequencing data

Sequencing companies normally report sequencing data as a FASTA file and also as ready nucleotide sequences via email. For sequence analysis, the following websites can be used:

http://blast.ncbi.nlm.nih.gov/Blast.cgi

http://xylian.igh.cnrs.fr/bin/align-guess.cgi

Here we will focus on the first website. On this website page, click on the “nucleotide blast” option (Figure  4 A). A new window opens. By default, the “blastn” (blast nucleotide sequences) option is marked (Figure  4 B). Then check the box behind “Align two or more sequences”. Now two boxes will appear. In the “Enter Query Sequence” box (the upper box), insert the desired sequence of your gene of interest, which is flanked by the restriction sites you have already designed for your PCR primers. In the “Enter Subject Sequence” box (the lower box), enter the sequence or upload the FASTA file you have received from the sequencing company. Then click the “BLAST” button at the bottom of the page. After a couple of seconds, the results will be shown on another page. A part of the alignment data is shown in Figure  4 C. For interpretation, the following points should be considered: 1) the number of identical nucleotides (shown under the “Identities” item) must be equal to the nucleotide number of your gene of interest. In our example, the number of nucleotides of the tdTomato gene together with those of the restriction enzyme sites and the Kozak sequence was 735. This equals the reported number (Figure  4 C). 2) The sequence identity (under the “Identities” item) should be 100%. Occasionally, the sequence identity is 100% but the number of identical nucleotides is lower than expected. This can happen if one or more of the initial nucleotides are absent. Remember, all sequencing technologies have an error rate. For Sanger sequencing, this error rate is reported to range from 0.001% to 1% [ 30 – 33 ]. Nucleotide substitution, deletion or insertion can be identified by analyzing the sequencing results [ 34 ]. Therefore, if the sequence identity does not reach 100%, the plasmid should be resequenced in order to differentiate errors of the PCR from simple sequencing errors. 3) Gaps (under the “Gaps” item) should not be present. If gaps occur, the plasmid should be resequenced.

Sequence analysis of the PCR product using the NCBI BLAST platform. (A) On the NCBI BLAST webpage, the “nucleotide blast” option is chosen (marked by the oval line). (B) The “blastn” option appears by default (marked by the circle). The sequence of the gene of interest (flanked by the restriction sites as previously designed for the PCR primers) and the PCR product are to be inserted to the “Enter Query Sequence” and “Enter Subject Sequence” boxes. Sequences can also be uploaded as FASTA files. (C) Nucleotide alignment of the first 60 nucleotides is shown. Two important items for sequence analysis are marked by oval lines.

The average length of a read, or read length, is at least 800 to 900 nucleotides for Sanger sequencing [ 35 ]. For the pJET vector one forward and one reverse primer need to be used for sequencing the complete gene. These primers can normally cover a gene size ranging up to 1800 bp. If the size of a gene is larger than 1800, an extra primer should be designed for each 800 extra nucleotides. Since reliable base calling does not start immediately after the primer, but about 45 to 55 nucleotides downstream of the primer [ 36 ], the next forward primer should be designed to start after about 700 nucleotides from the beginning of the gene. Different websites, including the following, can be used to design these primers:

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://www.yeastgenome.org/cgi-bin/web-primer

http://www.genscript.com/cgi-bin/tools/sequencing_primer_design

Being 735 bp in length, the size of the PCR product in this example was well within the range of the pJET sequencing primers.

After choosing the sequence-verified clone, vector and insert plasmids were digested by the Age I and Sal I restriction enzymes (Figure  5 ). This was followed by gel purification and ligation of the fragments. Transformation of competent E. coli with the ligation mixture yielded several clones that were screened by restriction enzymes. We assessed eight clones, all of which contained the tdTomato insert (Figure  6 ). It is important to pick clones that are large. Satellite clones might not have the right construct. We used a fast plasmid mini-preparation kit (Zymo Research) to extract the plasmid from 0.6 ml bacterial suspension. The yield and purity were satisfying for restriction enzyme-based screening (2.3 μg DNA; OD 260/280 = 1.82; OD 260/230 = 1.41). For large-scale plasmid purification, a maxi-preparation kit (QIAGEN) was used to extract the plasmid from 450 ml of bacterial culture (yield 787 μg DNA; OD 260/280 = 1.89; OD 260/230 = 2.22). The expected yield of a pBR322-derived plasmid isolation from 1.5 ml and 500 ml bacterial culture is about 2-5 μg and 500-4000 μg of DNA, respectively [ 37 ].

Vector and insert plasmid maps A) Illustration of the CloneJET plasmid containing the PCR product. Insertion of the PCR product in the cloning site of the plasmid disrupts the integrity of the toxic gene eco47IR and allows the growth of transgene positive clones. The plasmid was cut with the Age I and Sal I enzymes generating two fragments of 3 kb and 0.7 kb in size. The 0.7 kb fragment (tdTomato gene) was used as the insert for cloning. (B) Illustration of the vector plasmid. The plasmid was cut with the Age I and Sal I enzymes generating two fragments of 4.9 kb and 0.7 kb in size. The 4.9 kb fragment was used as the vector for cloning. AMP: Ampicillin resistance gene; PRE: posttranscriptional regulatory element; MPSV: myeloproliferative sarcoma virus promoter.

Screening of the final plasmid with restriction enzymes. Illustration of the final plasmid is shown. For screening, the plasmid was cut with the Bsiw I enzyme generating two fragments of 4.8 kb and 0.8 kb in size. AMP: Ampicillin resistance gene; PRE: posttranscriptional regulatory element; MPSV: myeloproliferative sarcoma virus promoter.

Some plasmids tend to recombine inside the bacterial host creating insertions, deletions and recombinations [ 38 ]. In these cases, using a recA-deficient E. coli can be useful (Table  1 ). Furthermore, if the GOI is toxic, incubation of bacteria at lower temperatures (25-30°C) and using ABLE C or ABLE K strains might circumvent the problem.

Viral production and transduction of target cells

To investigate the in vitro expression of the cloned gene, HEK293T cells were transfected with plasmids encoding the tdTomato gene, alpharetroviral Gag/Pol, and the vesicular stomatitis virus glycoprotein (VSVG) envelope. These cells, which are derived from human embryonic kidney, are easily cultured and readily transfected [ 39 ]. Therefore they are extensively used in biotechnology and gene therapy to generate viral particles. HEK293T cells require splitting every other day using warm medium. They should not reach 100% confluency for optimal results. To have good transfection efficiency, these cells need to be cultured for at least one week to have them in log phase. Transfection efficiency was 22%, as determined based on the expression of tdTomato by fluorescence microscopy 24 hours later (Figure  7 A-B). To generate a murine leukemia cell line expressing the tdTomato gene for immunotherapy studies, C1498 leukemic cells were transduced with freshly harvested virus (36 hours of transfection). Imaging studies (Figure  7 C) and flow cytometric analysis (Figure  7 D) four days after transduction confirmed the expression of tdTomato in the majority of the cells.

Assessing in vitro expression of the cloned gene. (A, B) HEK293T cells were transfected with Gag/Pol, VSVG, and tdTomato plasmids. The expression of the tdTomato gene was assessed using a fluorescence microscope. Fluorescent images were superimposed on a bright-field image for the differentiation of positively transduced cells. Transfection efficiency was determined based on the expression of tdTomato after 24 hours. Non-transfected HEK293T cells were used as controls (blue histogram). (C, D) The murine leukemia cell line C1498 was transduced with fresh virus. Four days later, transgene expression was assessed by fluorescence microscopy (C) and flow cytometry (D) . Non-transduced C1498 cells were used as controls (blue histogram). Scale bars represent 30 μm.

In this manuscript, we describe a simple and step-by-step protocol explaining how to exploit the power of PCR to clone a GOI into a vector for genetic engineering. Several PCR-based creative methods have been developed being extremely helpful for the generation of new nucleotide sequences. This includes equimolar expression of several proteins by linking their genes via a self-cleaving 2A sequence [ 40 , 41 ], engineering fusion proteins, as well as the use of linkers for the design of chimeric proteins [ 42 – 44 ]. Furthermore, protein tags [ 45 , 46 ] and mutagenesis (site-directed, deletions, insertions) [ 47 ] have widened the applications of biological engineering. The protocol explained in this manuscript covers for most situations of PCR-assisted cloning; however, alternative PCR-based methods are available being restriction enzyme and ligation independent [ 6 , 48 – 51 ]. They are of special interest in applications where restriction enzyme sites are lacking; nevertheless, these methods might need several rounds of PCR or occasionally a whole plasmid needs to be amplified. In such cases, the chance of PCR errors increases and necessitates sequencing of multiple clones. In conclusion, this guideline assembles a simple and straightforward protocol using resources that are tedious to collect on an individual basis thereby trying to minimize errors and pitfalls from the beginning.

Cell lines and media

The E. coli HB101 was used for the preparation of plasmid DNA. The bacteria were cultured in Luria-Bertani (LB) media. Human embryonic kidney (HEK) 293 T cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 mg/ml streptomycin, and 100 units/ml penicillin. A myeloid leukemia cell line C1498 [ 52 ], was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with the same reagents used for DMEM. Cells were split every other day to keep them on log phase.

Plasmids, primers, PCR and sequencing

A plasmid containing the coding sequence of the tdTomato gene, plasmid containing an alpha-retroviral vector, and plasmids containing codon-optimized alpharetroviral gag/pol [ 53 ] were kindly provided by Axel Schambach (MHH Hannover, Germany). A forward (5′- ACCGGTGCCACCATGGCCACAACCATGGTG-3′) and a reverse (5′-GTCGACTTACTTGTACAGCTCGTCCATGCC-3′) primer used for the amplification of the tdTomato gene were synthesized by Eurofins Genomics (Ebersberg, Germany).

The optimal buffers for enzymes or other reagents were provided by the manufacturers along with the corresponding enzymes or inside the kits. If available by the manufacturers, the pH and ingredients of buffers are mentioned. Primers were dissolved in ultrapure water at a stock concentration of 20 pmol/μl. The template plasmid was diluted in water at a stock concentration of 50 ng/μl. For PCR, the following reagents were mixed and filled up with water to a total volume of 50 μl: 1 μl plasmid DNA (1 ng/μl final concentration), 1.25 μl of each primer (0.5 pmol/μl final concentration for each primer), 1 μL dNTP (10 mM each), 10 μl of 5X Phusion HF buffer (1X buffer provides 1.5 mM MgCl2), and 0.5 μl Phusion DNA polymerase (2U/μl, Thermo Scientific).

PCR was performed using a peqSTAR thermocycler (PEQLAB Biotechnologie) at: 98°C for 3 minutes; 25 cycles at 98°C for 10 seconds, 66°C for 30 seconds, 72°C for 30 seconds; and 72°C for 10 minutes. To prepare a 0.8% agarose gel, 0.96 g agarose (CARL ROTH) was dissolved in 120 ml 1X TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH of 50X TAE: 8.4) and boiled for 4 minutes. Then 3 μl SafeView nucleic acid stain (NBS Biologicals) was added to the solution and the mixture was poured into a gel-casting tray.

DNA was mixed with 10 μl loading dye (6X) (Thermo Scientific) and loaded on the agarose gel (CARL ROTH) using 80 V for one hour in TAE buffer. The separated DNA fragments were visualized using an UV transilluminator (365 nm) and quickly cut to minimize the UV exposure. DNA was extracted from the gel slice using Zymoclean™ Gel DNA Recovery Kit (Zymo Research). The concentration of DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

For sequence validation, the PCR product was subcloned using CloneJET PCR cloning kit (Thermo Scientific). 1 μl of blunt vector (50 ng/μl), 50 ng/μl of the PCR product, and 10 μl of 2X reaction buffer (provided in the kit) were mixed and filled with water to a total volume of 20 μl. 1 μl of T4 DNA ligase (5 U/μl) was added to the mixture, mixed and incubated at room temperature for 30 minutes. For bacterial transfection, 10 μl of the mixture was mixed with 100 μl of HB101 E. coli competent cells and incubated on ice for 45 minutes. Then the mixture was heat-shocked (42°C/2 minutes), put on ice again (5 minutes), filled up with 1 ml LB medium and incubated in a thermomixer (Eppendorf) for 45 minutes/37°C/450RPM. Then the bacteria were spun down for 4 minutes. The pellet was cultured overnight at 37°C on an agarose Petri dish containing 100 μg/mL of Ampicillin. The day after, colonies were picked and cultured overnight in 3 ml LB containing 100 μg/mL of ampicillin.

After 16 hours (overnight), the plasmid was isolated from the cultured bacteria using the QIAprep spin miniprep kit (QIAGEN) according to the manufacturer’s instructions. 720 to 1200 ng of plasmid DNA in a total of 12 μl water were sent for sequencing (Seqlab) in Eppendorf tubes. The sequencing primers pJET1.2-forward (5′-CGACTCACTATAGGGAG-3′), and pJET1.2-reverse (5′-ATCGATTTTCCATGGCAG-3′), were generated by the Seqlab Company (Göttingen, Germany). An ABI 3730XL DNA analyzer was used by the Seqlab Company to sequence the plasmids applying the Sanger method. Sequence results were analyzed using NCBI Blast as explained in the Results and discussion section.

Manipulation of DNA fragments

For viewing plasmid maps, Clone Manager suite 6 software (SciEd) was used. Restriction endonuclease enzymes (Thermo Scientific) were used to cut plasmid DNA. 5 μg plasmid DNA, 2 μl buffer O (50 mM Tris–HCl (pH 7.5 at 37°C), 10 mM MgCl2, 100 mM NaCl, 0.1 mg/mL BSA, Thermo Scientific), 1 μl Sal I (10 U), and 1 μl AgeI (10 U) were mixed in a total of 20 μl water and incubated (37°C) overnight in an incubator to prevent evaporation and condensation of water under the tube lid. The next day, DNA was mixed with 4 μl loading dye (6X) (Thermo Scientific) and run on a 0.8% agarose gel at 80 V for one hour in TAE buffer. The agarose gel (120 ml) contained 3 μl SafeView nucleic acid stain (NBS Biologicals). The bands were visualized on a UV transilluminator (PEQLAB), using a wavelength of 365 nm, and quickly cut to minimize the UV damage. DNA was extracted from the gel slices using the Zymoclean™ gel DNA recovery kit (Zymo Research). The concentration of DNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific).

For the ligation of vector and insert fragments, a ligation calculator was designed (the Excel file available in the Additional file 1 ) for easy calculation of the required insert and vector volumes. The mathematical basis of the calculator is inserted into the excel spreadsheet. The size and concentration of the vector and insert fragments and the molar ratio of vector/insert (normally 1:3) must be provided for the calculation. Calculated amounts of insert (tdTomato) and vector (alpha-retroviral backbone) were mixed with 2 μl of 10X T4 ligase buffer (400 mM Tris–HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP (pH 7.8 at 25°C), Thermo Scientific), 1 μl of T4 ligase (5 U/μl, Thermo Scientific), filled up to 20 μl using ultrapure water and incubated overnight at 16°C. The day after, HB101 E. coli was transfected with the ligation mixture as mentioned above. The clones were picked and consecutively cultured for one day in LB medium containing ampicillin. Plasmid DNA was isolated using Zyppy™ plasmid miniprep kit (Zymo Research) and digested with proper restriction enzymes for screening. Digested plasmids were mixed with the loading dye and run on an agarose gel as mentioned above. The separated DNA fragments were visualized using a Gel Doc™ XR+ System (BIO-RAD) and analyzed by the Image Lab™ software (BIO-RAD). The positive clone was cultured overnight in 450 ml LB medium containing ampicillin. Plasmid DNA was isolated using QIAGEN plasmid maxi kit (QIAGEN), diluted in ultrapure water and stored at −20°C for later use.

Production of viral supernatant and transduction of cells

HEK293T cells were thawed, split every other day for one week and grown in log phase. The day before transfection, 3.5 × 10 6 cells were seeded into tissue culture dishes (60.1 cm 2 growth surface, TPP). The day after, the cells use to reach about 80% confluence. If over confluent, transfection efficiency decreases. The following plasmids were mixed in a total volume of 450 μl ultrapure water: codon-optimized alpharetroviral gag/pol (2.5 μg), VSVG envelope (1.5 μg), and the alpharetroviral vector containing the tdTomato gene (5 μg). Transfection was performed using calcium phosphate transfection kit (Sigma-Aldrich). 50 μl of 2.5 M CaCl 2 was added to the plasmid DNA and the mixture was briefly vortexed. Then, 0.5 ml of 2X HEPES buffered saline (provided in the kit) was added to a 15 ml conical tube and the calcium-DNA mixture was added dropwise via air bubbling and incubated for 20 minutes at room temperature. The medium of the HEK293T cells was first replaced with 8 ml fresh medium (DMEM containing FCS and supplement as mentioned above) containing 25 μM chloroquine. Consecutively the transfection mixture was added. Plates were gently swirled and incubated at 37°C. After 12 hours, the medium was replaced with 6 ml of fresh RPMI containing 10% FCS and supplements. Virus was harvested 36 hours after transfection, passed through a Millex-GP filter with 0.22 μm pore size (Millipore), and used freshly to transduce C1498 cells. Before transduction, 24 well plates were coated with retronectin (Takara, 280 μl/well) for 2 hours at room temperature. Then, retronectin was removed and frozen for later use (it can be re-used at least five times) and 300 μl of PBS containing 2.5% bovine serum albumin (BSA) was added to the wells for 30 minutes at room temperature. To transduce C1498 cells, 5 × 10 4 of cells were spun down and resuspended with 1 ml of fresh virus supernatant containing 4 μg/ml protamine sulfate. The BSA solution was removed from the prepared plates and plates were washed two times with 0.5 ml PBS. Then cells were added to the wells. Plates were centrifuged at 2000RPM/32°C/90 minutes. Fresh medium was added to the cells the day after.

Flow cytometry and fluorescence microscope

For flow cytometry assessment, cells were resuspended in PBS containing 0.5% BSA and 2 mM EDTA and were acquired by a BD FACSCanto™ (BD Biosciences) flow cytometer. Flow cytometry data were analyzed using FlowJo software (Tree Star). Imaging was performed with an Olympus IX71 fluorescent microscope equipped with a DP71 camera (Olympus). Images were analyzed with AxioVision software (Zeiss). Fluorescent images were superimposed on bright-field images using adobe Photoshop CS4 software (Adobe).

Abbreviations

Polymerase chain reaction

Gene of interest

Open reading frame

Melting temperature

Basic local alignment search tool

Vesicular stomatitis virus G glycoprotein

Luria-Bertani

Dulbecco’s Modified Eagle medium

Roswell Park Memorial Institute

Bovine serum albumin

Ethylenediaminetetraacetic acid

Fluorescence-activated cell sorting

Human embryonic kidney

Phosphate buffered saline

Fetal calf serum

Hydroxyethyl-piperazineethane-sulfonic acid

Ampicillin resistance gene

Posttranscriptional regulatory element

Myeloproliferative sarcoma virus promoter.

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Acknowledgments

The authors would like to thank Jessica Herbst, Abbas Behpajooh, Christian Kardinal and Juwita hübner for their fruitful discussions. We also thank Gang Xu for helping to design the cover page. This work was supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, the Deutsche Jose-Carreras Leukämiestiftung (grants SFB-738, IFB-TX CBT_6, DJCLS R 14/10 to M.G.S.) and the Ph.D. program Molecular Medicine of the Hannover Medical School.

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Sayed Shahabuddin Hoseini & Martin G Sauer

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SSH conceived the study subject, carried out experiments and drafted the initial manuscript. MGS participated in study design and coordination and edited the manuscript. Both authors have read and approved the final manuscript.

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13036_2014_161_moesm1_esm.xlsx.

Additional file 1: Ligation calculator. To calculate the amounts of the vector and insert fragments for a ligation reaction, you need to provide the size of the vector and insert (in base pairs), the molar ration of insert/vector (normally 3 to 5), vector amount (normally 50 to 100 ng), and vector and insert fragment concentrations (ng/μl). The computational basis of this ligation calculator is mentioned in the lower box. (XLSX 50 KB)

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Hoseini, S.S., Sauer, M.G. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering. J Biol Eng 9 , 2 (2015). https://doi.org/10.1186/1754-1611-9-2

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DOI : https://doi.org/10.1186/1754-1611-9-2

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• Polymerase chain reaction (PCR)
• Recombinant DNA
• Biological engineering
• Educational guide
• Transduction
• Transfection

Journal of Biological Engineering

ISSN: 1754-1611

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Quantitative polymerase chain reaction

Affiliation.

• 1 Division of Circadian and Visual Neuroscience, Univesity of Oxford, UK.
• PMID: 17417022
• DOI: 10.1007/978-1-59745-257-1_25

Quantitative PCR (qPCR) has entered widespread use with the increasing availability of real-time PCR. By the incorporation of fluorescent dyes in the reaction mixture, increases in amplification products can be monitored throughout the reaction, enabling measurements to be taken in the exponential phase of the reaction, before the reaction plateau. Whatever the platform or chemistry involved, the starting point of a real-time assay is a tissue-specific RNA and the end point of a real-time reaction is an amplification plot. As such, rather than focusing on specific platforms or chemistries, herein we address the basic principles that underlie sample preparation, experimental design, use of internal controls, assay considerations, and approaches to data analysis. The advent of real-time PCR has enabled high-throughput analysis of multiple transcripts from small tissue samples, with an unparalleled dynamic range and sensitivity. However, to new users, this technique may seem to require extensive optimization and troubleshooting to obtain reliable data; this is further compounded by the mass of technical variations present throughout the literature. The aim of this article is to provide the necessary basics to get a quantitative real-time PCR assay up and running, and to address some of the problems that may arise and how these may be resolved.

Publication types

• Research Support, Non-U.S. Gov't
• DNA Primers
• DNA, Complementary / genetics
• Data Interpretation, Statistical
• Deoxyribonucleases
• Fluorescent Dyes
• Polymerase Chain Reaction / instrumentation
• Polymerase Chain Reaction / methods*
• Polymerase Chain Reaction / statistics & numerical data
• RNA / genetics
• Reverse Transcription
• DNA, Complementary

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Polymerase chain reaction (pcr).

Nimrat Khehra ; Inderbir S. Padda ; Cathi J. Swift .

Affiliations

Last Update: March 6, 2023 .

• Introduction

The polymerase chain reaction (PCR) is a laboratory nucleic acid amplification technique used to denature and renature short segments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences using DNA polymerase I enzyme, an isolate from Thermus aquaticus, known as Taq DNA. [1] [2]  In 1985, PCR was introduced by Mullis and colleagues for which they received a Nobel prize. [3]  It is a monumental tool used in biomolecular sciences for its profound ability to examine and detect amplified components of DNA. [2]

PCR is a procedure that selectively focuses on a minuscule segment of DNA in a test tube. [1] [4]  Thermostability has the propensity to resist irreversible alterations in chemical and physical properties in extreme temperatures. [1]  Following several repetitive cycles of denaturation and renaturation in PCR procedures, Taq polymerase enzyme is preferred due to its heat-stable property, thus, allowing for the continuation of DNA synthesis despite the exposure of primers. [1] [2]  PCR has been the prominent procedure of choice in diagnosing a wide array of bacterial and viral infections, as well as screening genetic diseases due to its high sensitivity making it the gold standard testing procedure for numerous samples. [3]

• Testing Procedures

Polymerase chain reaction procedures begin with the collection of a small sample of DNA in a test tube. [4]  PCR consists of three major phases: denaturation, hybridization/annealing, elongation/amplification. [1]  During the denaturation phase, DNA is heated to 95 celsius (C) to dissociate the hydrogen bonds between complementary base pairs of the double-stranded DNA. [1]  Immediately following denaturation, the process of annealing occurs; annealing involves cooling the denatured DNA at a temperature ranging from 37-72 C allowing for the hydrogen bonds to reform. [4]  Annealing best occurs at temperatures between 55 C to 72 C. [1]

The specific temperature is determined based on the physical and chemical properties of the specific primers used in the solution. [3]  Primers are 20-25 nucleotides in length. [5]  Annealing allows for the primers to bind to the single-stranded DNA at their respective complementary sites beginning at the 3’ end of the DNA template. [1] [3]  Subsequently, the binding of the primers to their complementary sites on single-stranded DNA generates two double-stranded molecules. Finally, an optimal reaction temperature, 75-80 C, that is best suitable for enzyme-induced DNA replication is selected to ensure DNA polymerase activity. [3]

In order to initiate the functionality of DNA polymerase, double-stranded DNA is mandatory for the occurrence of replication. [3]  Thereafter, DNA polymerase synthesizes DNA in a 3’ to 5’ direction producing strands identical to the template strands. [3]  This procedure is repeated several times via a thermal cycler. [5]  A thermal cycler is a device that controls the time and temperature of each cycle and its respective steps. [5]  This ultimately leads to several duplicated DNA available in the tube. [1]

Following 30-40 cycles, repetitive cycles eventually taper off due to the limited capability of the reagent as well as other contributing factors such as accumulation of pyrophosphate molecules, excessive self-annealing, and the presence of PCR inhibitors in the sample. [3]  There are several inhibitors that can affect the proper functioning of PCR. The most common PCR inhibitors are proteinase K, phenol, Ethylenediaminetetraacetic acid (EDTA). [5]

Proteinase K has the propensity to break down Taq polymerase. [5]  Other substances that can negatively impact PCR tests are ionic detergents, heparin, spermidine, and hemoglobin. [5]  Additionally, bromophenol dyes and xylene cyanol can constitute complications in PCR testing. [5]  To overcome these issues, DNA templates can be cleansed by dialysis and precipitation by ethanol. Several other strategies to clean the DNA template include using chloroform for extraction purposes and chromatography. [5]

Following the aforementioned steps of PCR, the next step includes agarose gel electrophoresis using ethidium bromide. [1]  Subsequently, the gel is assessed in ultraviolet light. [1]  An imperative step in this component of the procedure requires examining the specificity of the results via transferring to a filter and implementation of a probe such as southern blot for hybridization. [1]  Lastly, it removes the amplification of primer dimers. [1]

There are various advantages of using PCR in basic and biomedical sciences. Over the years, it has acquired a renowned reputation making it the gold standard procedure for a multitude of reasons. [1]  Firstly, it is known for its ability to produce rapid results in a time-efficient manner; the PCR procedure typically requires a few hours to 3 days to generate results. [1]  A small sample of DNA or RNA (0.1- 5 mcg)  is necessitated to undergo this reaction. [1]  PCR also has the susceptibility to amplify  106 to 109 copies of DNA in a short period of time. [1]  PCR has the ability to generate efficient amplification products following cloning and expression due to the presence of restriction sites at terminal ends. [1]  

Real-time PCR

Real-time PCR is an alternative method to examine small segments of DNA  via the shortened duration of the cycles, elimination of post-PCR procedural handling steps, implementing fluorogenic labels as well as efficient detection of emissions. [6]  The discrete difference between real-time PCR and conventional PCR is the ability of real-time PCR to rapidly detect amplicons. [6]  The rapid detection of amplicons in real-time PCR is accomplished via surveillance by labeling primers, and fluorogenic molecules consisting of probes or amplicons. [6]

The disadvantage of real-time PCR in comparison to conventional PCR is that it requires opening the system to track the progression of amplicons. [6]  In addition, there are a few fluorogenic chemicals that are not compatible with the real-time PCR platforms. [6]  Lastly, it is more costly than conventional PCR. The aforementioned disadvantage of real-time PCR is predominantly due to the hardware incompatibilities and the accessibility of fluorogenic dyes. [6]

Reverse Transcriptase-PCR 

Reverse transcriptase-polymerase chain reaction (RT-PCR) is a procedure that uses mRNA for DNA amplification via DNA polymerase. [3]  The DNA polymerase in RT-PCR is expressed by retroviruses that consist of RNA eliciting complementary (cDNA). Conventional PCR and RT-PCR can be conjunctively used to study specific gene expressions from a qualitative standpoint. [3]

To date, Real-time PCR and RT-PCR are employed simultaneously to assess the quantitative difference in gene expression among various samples. [3] During the COVID-19 pandemic, RT-PCR has been the main diagnostic tool due to its high sensitivity, specificity, and rapidity. [7]  SARS-CoV-2 samples are generally acquired from various sites in the upper respiratory tract. [7]

Samples for PCR testing may be acquired from the nasopharynx, oropharynx, nostril, and oral cavity. [7]  The samples are collected via swabs, washes, and bronchoalveolar lavage. [7]

• Interfering Factors

There are a few drawbacks of polymerase chain reaction. This test is highly sensitive and has the susceptibility to detect the slightest contamination in DNA and RNA yielding inaccurate results. [3]  The primers designed for PCR require sequence to detect specific pathogens and genes. [3]  The occasional occurrence of non-specific annealing of primers to similar, but not exact, target genes is another interfering factor. [3]  The potential development of primer-dimers (PD) amplified by DNA polymerase can result in competition with PCR reagents. [3]

• Results, Reporting, and Critical Findings

In PCR, amplification of DNA can be observed with fluorescent dyes that bind to double-stranded DNA or probes that are sequence-specific. The amplification reaction consists of a quantification cycle, Cq. Cq is described as the number of fractional cycles necessitated for the fluorescence to meet the preset for quantification. [8]

After determining Cq, a qualitative conclusion can be deduced, or a quantitative analysis may be further conducted. Cq is dependent on PCR efficiency; PCR efficiency involves assessment of the amplification efficiency which is explained as fold increase/cycle with a fold value ranging from 1 to 2, with a fold value of 2 indicating 100% PCR efficiency. PCR efficiency is derived from standard curves and amplification curves. [8]

Standard curve PCR efficiency increases the likelihood of dilution errors ultimately affecting the accurate quantification of several clinical and biological samples. However, individual amplification curves do not include confounding variables for the analysis of PCR efficiency resulting in varying results compared to standard curves for the same assay. Accurate computation of the target quantity is essential for appropriate amplification efficiency that will be reflected in the analysis. [8]

A low PCR efficiency requires additional cycles to reach an appropriate quantification threshold resulting in a higher Cq. The presence of amplification following the utilization of a valid probe-based assay is indicative of the sample containing the particular target and subsequently concluding it as diagnostically positive. Due to the Poisson variation, the lack of amplification is not a valid criterion to classify a reaction as negative. [8]

As mentioned earlier, qPCR measures DNA or RNA in various diagnostic and biological samples via the Cq. qPCR is often computed with the assumption that all assays are 100% efficient. Additionally, reporting of qPCR involves Cq, delta-Cq, or delta-delta-Cq. For significant and purposeful interpretation of biological, clinical, and diagnostic samples, efficiency corrected should be utilized in qPCR testing. Thus, it is essential to consider these factors when interpreting and reporting PCR efficiency to yield adequate results. [8]

The stage and degree of a patient's ailment can be reckoned with by utilizing cycle threshold (Ct) values concurrently with clinical manifestations and disease history. Moreover, healthcare professionals can further surveillance the progression of diseases and foreshadow steps to recover and resolve ailment by repeating the PCR test and generating serial Ct values. Ct values can also aid contact tracers to focus on patients with a more elevated viral genomic load, signifying a higher risk for disease transmission. [9]

• Clinical Significance

PCR is used in basic and biomedical sciences which has substantial laboratory and clinical significance due to its high sensitivity, specificity, and rapidity. [1] [4] [10]  It has been frequently used to recognize various viral infectious disease microorganisms. Some of the viral pathogens detected via PCR include human papillomavirus (HPV), human immunodeficiency virus (HIV), herpes simplex virus (HSV), SARS-CoV-2, varicella-zoster virus (VZV), enterovirus, cytomegalovirus (CMV), and hepatitis B (Hep-B), hepatitis C (Hep-C), hepatitis D (Hep-D), and hepatitis E (Hep-E).  [1] [3] [11] [7]  The presence of bacterial, fungal, parasitic organisms and various immunodeficiencies can be detected via PCR making it an instrumental tool in clinical diagnoses and practice. [1]

Rapid detection of microbial pathogens via rapid real-time PCR allows for clinicians to promptly administer tailored treatment thus, reducing hospitalizations, preventing inappropriate administration of antibiotics, in turn, antibiotic resistance. [6]  Real-time PCR has the propensity to detect specific bacterial species such as Mycobacterium species, leptospira genospecies, chlamydia species, Legionella pneumophila, Listeria monocytogenes, and Neisseria meningitides. [6]  Real-time PCR has also proven to effectively detect and examine antibiotic-resistant strains such as staphylococcus aureus, staphylococcus epidermidis, helicobacter pylori, and enterococcus. [6]  Furthermore, fulminant diseases are also detected and examined early due to the high sensitivity, specificity, and rapidity of the real-time PCR test making it the ideal procedure for medical conditions such as meningitis, sepsis, and inflammatory bowel diseases (IBD). [6]

Additional microbial pathogens that are infamous for causing food-borne related illnesses such as group B streptococci, mycobacterium species, Bacteroides vulgatus, and escherichia coli (e.coli) can also be identified via real-time PCR testing. [6]  Due to the rapid nature of real time-PCR tests, earlier detection can aid in tracing its source, in turn, controlling existing and potential outbreaks. [6]  Fungal, parasitic, and protozoan pathogens have also been identified on real-time PCR testing such as aspergillus fumigatus and aspergillus flavus, cryptosporidium parvum, and toxoplasma gondii.  [6]

PCR is also used to study the histopathology of various viral and cellular genes to comprehend and diagnose malignant diseases in humans. [1]  Additionally, PCR has been used in analyzing forensic samples, point mutations, DNA sequencing, in vitro mutagenesis. [1] [5]  It has a rapid propensity to screen and detect specific alleles ideal in prenatal genetic testing for carrier status. [3]  PCR also has the ability to detect the presence of disease and mutations in utero and in adults. [12]  

• Quality Control and Lab Safety

Contamination of PCR

Conventional PCR is the gold standard for screening and detecting a wide scope of scientific areas of interest due to its promising results. Adequate handling following PCR procedure is imperative for proper assessment of amplicon. [6]  However, in conventional PCR, post-procedural improper handling can lead to the proliferation of amplicon within the laboratories. [6]

To prevent contamination of PCR, it is crucial to have a designated area of the laboratory exclusive for PCR testing to limit unnecessary turbulence within the area. [5]  Face masks, gloves, and hair caps should be worn at all times in the laboratory to prevent the occurrence of contamination. [5]  Preparation and storage of solutions in equipment such as pipettes, glassware, and plasticware should not be contaminated or exposed with DNA. [5]

A specific section in a freezer, that is closest to the laminar flow hood, should contain enzymes and buffers. [5]  Use of any reagents should be discarded immediately. [5]   A laminar flow hood with ultraviolet (UV) lights is the ideal location in a laboratory to perform PCR. [10] Equipment such as pipettes, sterile gloves, and microcentrifuge should be present within the laminar flow hood. [5]

Automatic pipettes are known to cause contamination, thus, positive displacement pipettes should be employed for proper handling of the reagents. [5]  Any laboratory equipment that is disposable such as pipette tips and tubes should not be autoclaved prior to use. [5]  Additionally, disposable equipment should be used directly from its respective packaging. [5]

Prior to using microcentrifuge tubes consisting of reagents exclusively for PCR, centrifugation is necessitated for approximately 10 seconds to allow the fluid to settle at the bottom of the tube preventing the occurrence of contamination. [5]  Post amplification techniques should be completed at a laboratory bench as opposed to the designated area for PCR testing. [5]

• Enhancing Healthcare Team Outcomes

Efficient use of PCR by the interprofessional healthcare team can lead to early detection of bacterial and viral pathogens prompting earlier treatments. This can also further aid in preventing antibiotic resistance and viral outbreaks, respectively. The interprofessional health care team comprises a primary care physician, pathologist, infectious disease specialist, lab technician, and nurses.

The polymerase chain reaction is a nucleic acid amplification testing procedure that consists of denaturing, renaturing, elongating, and amplifying a short segment of DNA or RNA. This is implemented by incorporating DNA I polymerase, which is derived from Thermus aquaticus, also known as Taq polymerase. Taq polymerase consists of thermostable properties preventing the irreversible alteration of the DNA or RNA physical and chemical properties, making it ideal for the highly sensitive polymerase chain reaction procedure for diagnosing a wide range of bacterial and viral infections, as well as screening genetic diseases.

Laboratory technicians should be fully trained in the safe handling and use of samples to ensure quality and prevent contamination. Face masks, gloves, and hair caps should be worn at all times in the laboratory. Storage of solutions in their respective equipment (pipettes, glassware, plasticware) should be done with caution to prevent DNA from being exposed and contaminated. The interprofessional team of healthcare providers should be up to date with the latest guidelines and management strategies for patients with confirmed communicable diseases.

This integrated team-based approach provides care coordination from all interprofessional team members to further advance the health of patients suffering from infectious diseases. Patients should also be thoroughly informed on laboratory findings and counseled on preventative measures, and the importance of medication compliance is needed. Patients should also be educated on disease transmission and preventive measures they can incorporate to ensure public health and safety. Continuous communication between the healthcare team and their patients can help form a therapeutic alliance to prevent complications and spread of communicable disease, ensure patient and public safety, and preserve the quality of life.

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Disclosure: Nimrat Khehra declares no relevant financial relationships with ineligible companies.

Disclosure: Inderbir Padda declares no relevant financial relationships with ineligible companies.

Disclosure: Cathi Swift declares no relevant financial relationships with ineligible companies.

This book is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ), which permits others to distribute the work, provided that the article is not altered or used commercially. You are not required to obtain permission to distribute this article, provided that you credit the author and journal.

• Cite this Page Khehra N, Padda IS, Swift CJ. Polymerase Chain Reaction (PCR) [Updated 2023 Mar 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-.

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