 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. Poster PCR Poster RT-PCR Poster Boards PCR movies Roche Applied Science - PCR Application Manual 3rd Edition
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.
What is the polymerase chain reaction (PCR)?
"PCR
has
transformed molecular biology through vastly
extending the capacity to identify, manipulate
Paul Rabinow, Making
PCR, A Story of Biotechnology, University of Chicago Press,
1996
assays are putting quantitative PCR back in the spotlight by Elizabeth Zubritsky http://pubs.acs.org/hotartcl/ac/99/mar/pcr.html Analytical Chemistry News & Features, March 1, 1999; pp.
191A-195A Classical block PCR & RT-PCR Real-time PCR
vs. traditional block PCR This tutorial
will discuss
the evolution of traditional PCR methods towards
Standard PCR Protocol
by Ed Rybicki , January 1994, February 2001
This is dealt with under the following subheadings:
by Applied Biosystems http://www.appliedbiosystems.com/support/tutorials/pcropt/ 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.
University of
Salzburg, Institute of Chemistry and Biochemistry, 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.
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 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.
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