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
COMMENTS
The advent of the polymerase chain reaction (PCR) radically transformed biological science from the time it was first discovered (Mullis, 1990). For the first time, it allowed for specific detection and production of large amounts of DNA. PCR-based strategies have propelled huge scientific endeavors such as the Human Genome Project.
The polymerase chain reaction (PCR) is a technique in molecular biology to amplify a single or a few. copies of a piece of DNA across several orders of magnitude, generating thousands to millions ...
Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR — detection and expression analysis of gene(s) in real-time — has revolutionized the 21 st century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to ...
2. Basic Principles. Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR, is a modification of the PCR strategy which allows monitoring of the PCR progress in real-time PCR itself is an enzymatic process used in vitro for the amplification of a selected DNA region through several orders of magnitude, generating thousands to millions of copies of a specific DNA ...
Abstract. DNA-based procedures are becoming increasingly common within the analytical laboratory where the polymerase chain reaction (PCR) has become an indispensable technique. Developed in 1985 by Kary B. Mullis, PCR revolutionized the way that deoxyribonucleic acid (DNA) could be copied. Mullis's invention allowed researchers to make ...
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. ... Research Open Access 10 Apr ...
From gene expression studies to identifying microbes, quantitative polymerase chain reaction (qPCR) is widely used in research and medical diagnostics. In transmittable diseases like the Ebola outbreak in West Africa (2014-2016), or the present SARS-CoV2 pandemic qPCR plays a key role in the detection of infected patients. Although the technique itself is decades old with reliable approaches ...
basic molecular research right through to an approved molecular diagnostic assay. The exploration of the current wide range of applications of the real-time PCR method is critical, including its feasibility in low-middle income countries. 2. Basic Principles Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR,
Over nearly 30 years, quantitative polymerase chain reaction (qPCR) has developed into an essential tool. It allows researchers to amplify and quantify the presence of specific DNA sequences.
The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA ...
The development of the PCR system and its applications. (A) A prototype PCR thermal cycler developed by Cetus Corporation in 1986, which is the first model embedding the software cycling controller in the thermal cycling block.(B) Space-domain PCR.(C) Time-domain PCR.(D) Use of anchored allele-specific probes and labeled primers for the colorimetric detection of mutations in the HBB gene.
Background 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 polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to ...
This essay on the polymerase chain reaction is one of a series developed as part of FASEB'S efforts to educate the general public, and the legislators whom it elects, about the benefits of fundamental biomedical research—particularly how investment in such research leads to scientific progress, improved health, and economic well-being. 1 ...
Abstract. Kary Mullis developed a biochemical technology Called Polymerase Chain Reaction (PCR) which can be used to amplify a single copy or a few copies of a piece of DNA across several orders ...
Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region ...
The present paper is an attempt to review basics of PCR, a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copy of a particular DNA sequence. The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of ...
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 ...
Abstract. 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 ...
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs ...
Polymerase chain reaction (PCR) is a robust technique to selectively amplify a specific segment of DNA in vitro . [ 1] PCR is performed on thermocycler and it involves three main steps: (1) denaturation of dsDNA template at 92-95°C, (2) annealing of primers at 50-70°C, and (3) extension of dsDNA molecules at approx. 72°C.
Decaying Dark Matter: Modern Cosmology and the Dark Matter Problem.D. W. Sciama. Cambridge University Press, New York, 1994. xviii, 216 pp., illus. Paper, $29.95 or ...
Feature papers represent the most advanced research with significant potential for high impact in the field. A Feature Paper should be a substantial original Article that involves several techniques or approaches, provides an outlook for future research directions and describes possible research applications. ... Lorenz, T.C. Polymerase Chain ...
INTRODUCTION. The development of molecular biology was one of the greatest achievements in biological science in the century XX. The discovery of Polymerase Chain Reaction (PCR) brought enormous benefits and scientific developments such as genome sequencing, gene expressions in recombinant systems, the study of molecular genetic analyses, including the rapid determination of both paternity and ...