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Ensuring Successful PCR Using Online Resources

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LabFact #2

RT-PCR should be performed in the absence as well as presence of the reverse transcriptase, to assess DNA contamination in the template RNA. In addition, "no-template" negative control reactions should always be performed.

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Abstract

PCR is a technique widely used to amplify a single template DNA and generate many copies, and has had a profound effect on biological research and human identity. Here we discuss the many resources available on the Web that cover everything from learning the basics of PCR to troubleshooting problems.

Anupama Gopalakrishnan

Promega Corporation
Publication Date: 2009

Introduction

In 1985, a relatively simple technique was developed to amplify sequence-specific DNA fragments in vitro. Called polymerase chain reaction (PCR), this method was able to multiply a small number of DNA molecules, resulting in many copies of the template. To accomplish this, the DNA is mixed with a thermostable DNA polymerase, deoxyribonucleotides, primers specific to the target DNA and reaction buffer, and this mixture is heated to denature the double-stranded DNA template, then cooled to allow the primers to bind to the template and Taq DNA polymerase to synthesize the full DNA strand before heating the mixture again. By repeating the heating and cooling cycles, a single molecule of DNA can be amplified exponentially to produce tens, hundreds, thousands or millions of copies, all identical to the original template.

The implications of this DNA amplification technique took many years to realize, but now the word “PCR” is present in the vernacular, discussed on television and in movies and performed by millions of thermal cycling instruments in laboratories the world over. PCR technology has proven extraordinarily versatile, revolutionizing modern biology by accelerating discoveries in a vast range of scientific disciplines, including genomics, clinical diagnostics, forensic sciences, industrial quality control and environmental sciences. Prominent examples of how PCR facilitates scientific progress include the mapping of the human genome, single-sperm analysis, disease diagnosis, genotyping, molecular archaeology and drug discovery.

PCR has opened up a wide range of applications, including reverse transcriptase PCR (RT-PCR) to detect transcripts in cells and quantitative real-time PCR (qPCR) to determine the copy number of the template. Quantitative real-time PCR is used to determine changes in gene expression during development, drug treatment and disease conditions, helping scientists better understand various biological processes.

Given the influence of this technology in molecular biology, it is imperative that PCR reagents and conditions be optimized to ensure high specificity and sensitivity. It is equally important to have easy access to tools and resources that are helpful in planning, optimizing and troubleshooting PCR experiments. A leading provider of both PCR reagents and technical information, Promega offers many helpful resources for anyone using this technology.

Promega Resources Available on the Web

PCR Basics

Both students and educators will find the animation illustrating the scientific basis of PCR useful, but there are also tools instructors can use in the classroom to discuss PCR. For more detailed information about PCR, thermostable DNA polymerases, PCR protocols, troubleshooting amplification reactions and more, the online Protocols and Application Guide provides a detailed and comprehensive chapter.

The Right PCR Enzyme

With a bewildering array of thermostable DNA polymerase choices, templates and features that enhance PCR, it can be difficult to determine which enzyme or system best suits your needs. The Amplification Product Selector helps the researcher choose the right amplification kit based on starting sample type, fidelity requirements, downstream processing and ease of reaction setup.

The routine PCRhot-start PCR and reverse transcription PCR (RT-PCR) Web pages provide information about individual components or ready-to-use master mixes (buffer, nucleotides and enzyme already premixed), thereby allowing researchers to select the product best suited to their experimental needs. While a kit containing individual components is useful to optimize PCR conditions such as adjusting magnesium concentration, master mixes provide convenience and better data reproducibility once the optimal PCR conditions are established.

A proofreading high-fidelity thermostable DNA enzyme such as Pfu DNA polymerase or Tli DNA polymerase produces blunt-ended PCR products due to its 3´→5´ exonuclease activity, while a nonproofreading enzyme like GoTaq® DNA Polymerase or GoTaq® Hot Start Polymerase leaves 3´ adenosine overhangs, which facilitate easy cloning intoT vectors, like the pGEM®-T and pGEM®-T Easy Vectors, which have a 3´ thymidine overhang. Therefore, identifying the characteristics of different thermostable polymerases helps select the right enzyme for PCR. An additional benefit of using GoTaq® DNA polymerase: the supplied Green Reaction Buffers allow the user to go from amplification directly to gel analysis due to the presence of blue and yellow gel loading dyes. TheGoTaq® Green Master Mix features the GoTaq® DNA Polymerase with the convenience of direct gel loading with fewer manipulations and less likelihood of contamination.

PCR Optimization

Some reactions require optimization of reaction components for maximum specificity and sensitivity. The GoTaq® Flexi DNA Polymerase gives the researcher flexibility to titrate enzyme, template, primer, nucleotides and magnesium, which results in a reproducible, single PCR product when reaction conditions are optimized. As a required cofactor for thermostable DNA polymerases, magnesium can greatly affect amplification success. Template DNA concentration, chelating agents present in the sample (e.g., EDTA or citrate), dNTP concentration and the presence of proteins can all affect the amount of free magnesium in the reaction. While Taq DNA polymerase is inactive in the absence of adequate free magnesium, excess free magnesium reduces enzyme fidelity and may increase the level of nonspecific amplification.

One of the most frequently encountered PCR problems is the appearance of nonspecific bands caused by false priming. Aside from optimizing the entire amplification reaction, two relatively simple solutions exist: adjust the primer annealing temperature and use a hot-start DNA polymerase.

  • Using the BioMath Calculator before programming a thermal cycler can improve specificity and reduce nonspecific bands by determining the melting temperature (Tm) of the primer and %GC content, which is very useful when choosing the right annealing temperature. You can also select Promega primers to examine their Tm and determine the Tm of your primers in different reaction buffers.
  • Promega GoTaq® Hot Start Polymerase allows hot-start PCR and provides greater specificity, yield and sensitivity by reducing nonspecific priming and primer-dimer formation by minimizing polymerase activity until the reaction is heated to a temperature that favors specific priming. Hot-start technology is described in a Promega Notes article describing the flexibility of room-temperature setup and a short initial denaturation to restore polymerase activity. Assembled reactions can sit at room temperature for up to 24 hours before starting the thermal-cycling procedure. This benefit is important for scientists using high-throughput or robotic platforms. If you prefer the convenience of master mixes with the benefits of a hot-start enzyme, the GoTaq® Hot Start Green or Colorless Master Mixes can be used.

Never underestimate the importance of PCR controls. The GoTaq® PCR Core System II provides positive control DNA and primers to ensure all reaction components are working well. When troubleshooting problems with PCR, a no-template control is a useful tool for diagnosis.

PCR Products in Downstream Applications

Once the desired product is amplified, it can be used for research, clinical or forensics analysis. Some applications use the PCR product directly; others need purified DNA. Articles in the PubHub discuss PCR optimization, purification of PCR products and compatibility with various downstream applications including: restriction enzyme digestion, cell-free protein expression (e.g., TnT® T7 Quick for PCR DNA), T-vector cloning, directional cloning and expression using theFlexi® Vectors and designing appropriate PCR primers with the Flexi® Vector Primer Design Tool, The Citations database highlights peer-reviewed papers discussing the use of various products and provide customer-modified protocols for specific applications, thereby eliminating the need to reoptimize the experiments.

Quantitative PCR (qPCR) Tools

Resources for quantitative real-time PCR begin by illustrating the basic principle of the Plexor® reaction. The Plexor® Systems measure a reduction in fluorescent signal during amplification (Figure 1). Amplification uses only two primers, one of which contains both a fluorescent tag and a modified base. As amplification proceeds, fluorescence is reduced by site-specific incorporation of a fluorescent quencher inserted opposite the complementary modified base. Multiplex assay design is simplified by using the software for Plexor® primer design and data analysis.

Figure 1. Representation of the Plexor® real-time PCR process.

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The Plexor® technology has been adopted by DNA-typing labs to quantify DNA. Promega offers the Plexor® HY System, a real-time PCR assay specifically designed to determine the concentration of total human DNA and male human DNA simultaneously in a single reaction. The data can be analyzed using free software available for download on the Promega Web site (registration required). This system quantitates DNA prior to multiplex short tandem repeat (STR) amplification, a method used for DNA typing, including paternity testing, forensic DNA analysis and human identity testing.

Forensic Applications

The PowerPlex® 16 System is one example of an STR amplification system that coamplifies and fluorescently labels 16 highly polymorphic STR loci. In addition to protocols, locus information and FAQs, the Genetic Identity Web page also contains resources to facilitate automatic labeling of STR data generated by the PowerPlex® Systems. Supplemental information on DNA typing and population statistics helps the various forensic agencies interpret their data by providing information about allele frequencies of various loci in different populations.

Summary

Promega provides researchers with reagents, tools and other resources to facilitate rapid and convenient experimental setup for PCR applications. From animations describing the basics of PCR and quantitative PCR and advice on selecting the right polymerase and primer melting temperature to data analysis software for interpreting complex quantitative RT-PCR data, Promega offers technical assistance and resources at every step for scientists of different levels of expertise to plan and perform their experiments.

How to Cite This Article

Gopalakrishnan, A. Ensuring Successful PCR Using Online Resources. [Internet] 2009. [cited: year, month, date]. Available from: http://www.promega.com/resources/pubhub/enotes/ensuring-successful-pcr-using-online-resources/

Gopalakrishnan, A. Ensuring Successful PCR Using Online Resources. Promega Corporation Web site. http://www.promega.com/resources/pubhub/enotes/ensuring-successful-pcr-using-online-resources/ Updated 2009. Accessed Month Day, Year.

Products may be covered by pending or issued patents or may have certain limitations on use. Please visit our patent and trademark web page for more information.

Apple and iPod are trademarks of Apple Inc., registered in the U.S. and other countries. iPhone is a trademark of Apple Inc. SYBR is a registered trademark of Molecular Probes, Inc. TaqMan is a registered trademark of Roche Molecular Systems, Inc.

Figures

Figure 1. Representation of the Plexor® real-time PCR process.

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