Polymerase Chain Reaction (PCR)
Reverse Transcription - Polymerase Chain Reaction (RT-PCR)

The Nobel Prize in Chemistry 1993

Polymerase Chain Reaction - The PCR method – a copying machine for DNA molecules

DNA molecules can be mass-produced from incredibly small amounts of material with PCR. Kary Mullis' discovery allows the chemist to mimic the cell's own natural DNA replication process in a test tube. It has now become much easier to characterise and compare the genetic material from different individuals and organisms.

PCR and RT-PCR are techniques so common in today's laboratories that it is easy to forget its revolutionary impact, enables the specific amplification and detection of as little as a single copy of a particular nucleotide sequence. However, PCR has the potential to be used not just for the detection of specific sequences, but also for their quantification, because of the quantitative relationship between the amount of starting target sequence and the amount of PCR product at any given cycle that falls within the reaction's exponential range.

Making  PCR  (Polymerase Chain Reaction)

What is the polymerase chain reaction (PCR)? 
It is the supreme biotechnological invention.

  "PCR has transformed molecular biology through vastly extending the capacity to identify, manipulate 
and reproduce DNA. It makes abundant what was once scarce - the genetic material required for experimentations."

                                           Paul Rabinow, Making PCR, A Story of Biotechnology, University of Chicago Press, 1996

Widespread interest in gene quantitation and high-throughput 
assays are putting quantitative PCR back in the spotlight

   by Elizabeth Zubritsky


Analytical Chemistry News & Features, March 1, 1999; pp. 191A-195A

Classical block  PCR   &  RT-PCR

Real-time PCR    vs.   traditional block PCR
by ABI Applied Biosystems

This tutorial will discuss the evolution of traditional PCR methods towards
the use of Real-Time chemistry and instrumentation for accurate quantitation. 

Standard PCR Protocol 

           by   Ed Rybicki , January 1994, February 2001
in   Molecular Biology Techniques Manual    Third Edition
 Edited by:  Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki


This is dealt with under the following subheadings:

PCR  Optimization:  Reaction Conditions and Components

by Applied Biosystems


The GeneAmp PCR process is widely employed in a tremendous variety of experimental applications to produce high yields of specific DNA target sequences. Since no single set of conditions can be applied to all PCR amplifications, individual reaction component concentrations (and time and temperature parameters) must be adjusted within suggested ranges for efficient amplification of specific targets. While there are a number of possible concentration parameters, logical titrations of interrelated reaction components can be readily defined. In addition, the time and temperature optima can often be determined within a few experiments.

The PCR plateau phase - towards an understanding of its limitations

by  Kainz P.
Biochim Biophys Acta  2000 Nov 15;1494(1-2):23-27

University of Salzburg, Institute of Chemistry and Biochemistry,
Hellbrunnerstrasse 34, A-5020, Salzburg, Austria.

The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. A 30-fold molar excess of fragments to polymerase molecules was found to be required for a complete inhibition of Taq, Tfl and Pwo DNA polymerase. This stoichiometric relation remained constant when PCR amplifications were performed using polymerase concentrations of 0.5, 1 or 1.5 U/50 microl reaction volume. The amount of 1 kb DNA fragments required for a complete inhibition was similar to the product yield of the controls (no fragment added), that were run to plateau phase levels. Additionally, PCR mixtures, that were subjected to different numbers of cycles, were compared in their ability to extend 3'-recessed ends by using a hairpin extension assay. The presence of endogenous amplicon DNA accumulated in later PCR cycles was found to inhibit completely the activity of DNA polymerase. PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products. 

PCR Optimization: Building The Perfect Beast

  by Debra Swanson  The Scientist 13 [4]: 26, Feb. 15, 1999

During the past decade, the polymerase chain reaction has become one of the most versatile tools used in
the molecular biology laboratory. Applications range from the analysis of genomic DNA samples and the production of DNA fragments for cloning systems to direct DNA sequencing. The reaction requires a minimum of reagents and equipment compared to many lab protocols and can be set up simply and quickly. It can be as useful to the solo underfunded bench scientist as it is to teams of production scientists using the most advanced automated equipment, generating significant amounts of data in a short time. 

   The basic premise of the reaction is simple--DNA can be amplified many thousandfold or millionfold in a tube. A typical amplification reaction includes a minimal amount of the nucleic acid template, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium, optional extras, and a thermostable DNA polymerase. The components are mixed and put into an automated thermal cycler that cycles the reaction at different temperatures for varying amounts of time over and over, directing the reaction to denaturation, primer annealing, or elongation of the product. This product can be analyzed and quantified or used in other scientific endeavors. Utilization of DNA amplification has crossed over into disciplines outside of the common realm of molecular biology into botany, forensics, evolutionary science, and biotechnology. 

   So if PCR is such a wonderfully simple, fast, and reliable technique that produces a mountain of results, what's the "catch"? What is it about PCR that can elate us or deflate us? The "catch" is that the reaction can be a beast to optimize, resulting in hours of frustration and futility in the lab. "PCR enhancement" can describe any number of manipulations of the reaction that optimize specificity and yield of the amplification. Enhancers may be    new and expensive marketed reagents, or quite simply, tricks of the trade. Whatever its origin, an enhancement will become a necessity, and perhaps a welcome relief, in every PCR-oriented laboratory at some point. 

PCR and multiplex PCR: guide and troubleshooting

 by   OCTAVIAN HENEGARIU,  Yale University


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