Download Polymerase Chain Reaction (PCR) Techniques

A Technical Note...
[
Polymer ase Chain Reaction
(PCR) Techniques
ClinicalRM
In 1993, Dr. Kary Mullis received the Nobel Prize in Chemistry, “for his
invention of the polymerase chain reaction (PCR) method.” With this simple
notation, the Noble Prize selection committee acknowledged one of the most
important advances in molecular biology. In this short article, let us describe
what the process of PCR entails.
personnel
are on the
]
PCR is a technique used to amplify a single, or a few copies of, a piece of
DNA (or in some cases RNA), generating thousands to millions of copies
of the particular DNA sequence. PCR relies on thermal cycling, which
consists of repeated cycles of heating and cooling the reaction involving DNA. Primers, or short DNA
fragments, containing complementary sequences to the “target” region, along with a DNA polymerase,
are the important components to enable very selective and repeated amplification of the “target” DNA.
As the PCR progresses, the DNA that is resulting from the PCR process is itself used as a template
for the reaction progression. The process continues not unlike a nuclear chain reaction – a polymerase
chain reaction – resulting in an exponential amplification of the original “target” DNA. PCR is a very
flexible process and can be modified to provide a wide array of genetic transformations.
Also read our tecnical notes on related
topics:
• Nanotechnology
Most PCR techniques use heat-stable DNA polymerase, often Tag-polymerase, an enzyme that was
isolated from Thermus aquaticus – a thermophylic bacterium. The DNA polymerase is an enzyme that
assembles a new DNA strand from nucleotides using a single strand of DNA as a template and DNA
oligonucleotides (Primers) required for the DNA synthesis. Most PCR methods use this thermal
cycling to physically separate the two DNA strands at a high temperature (DNA melting). At the lower
temperature, each strand is used as a template to copy its counterpart sequence (in the presence of
DNA polymerase). This extremely powerful process of heating and cooling continues to amplify the
target sequence. • Resistant Pathogens
■ Jere Boyer, Ph.D.
cutting edge
of PCR research.
• Alternatives to Antibiotic Use
Using PCR Techniques in the Field...
References
▶ http://faculty.plattsburgh.edu/
donald.slish/PCR.html
▶ http://www.accessexcellence.
org/RC/CT/polymerase_chain_
reaction.php
ClinicalRM personnel are on the cutting edge of PCR research. ClinicalRM employees have used most
of the research platforms available in the modern biomedical research laboratory. Among the most used
platforms are those involved with polymerase chain reactions (PCR). Recently, outbreaks of resistant
organisms, such as Acinetobacter baumannii, has required researchers, including ClinicalRM personnel, to
use genomic techniques like PCR to determine the spread of these highly resistant isolates.
In the area of wound infections, ClinicalRM personnel have been involved in the use of Q-PCR with
species specific Primers to amplify genes present from a total DNA extract from wound sites. In
conjunction with standard controls, it is possible to accurately determine a quantitative measurement
of bacterial load. Studies involving ClinicalRM personnel are also underway utilizing RT-PCR to
detect potential contamination of hospital surfaces by bacteria of clinical relevance. Such information
is vital in detecting the potential spread of organisms in the clinical environment. Similar studies are
underway in the public arena as well.
▶ http://www.dnalc.org
▶ http://www.answers.com/topic/
Our personnel are also involved in the development of new PCR platforms. Recently, ClinicalRM
employees, along with our industry and Government partners, have developed disposable PCR kits that
can be used to detect organisms, or the exposure to organisms, in hostile environments.
▶ http://www.pcr-encyclopedia.
Applications & Literature
polymerase-chain-reaction
com
▶ http://www.cepheid.com
▶ http://www.warmwell.com/
rapid.html
▶ Higgins, P.G. et al. (2007).
Clinical. Microbiol. & Infection
13:1199-1201.
There are at least 21 variations of the basic PCR process and we cannot discuss them all in this short
article. One of the more popular variations is Reverse Transcription, or RT-PCR, which produces
cDNA from RNA and is often used for determination of expression of a gene or to identify the
sequence of an RNA transcript. If the genomic DNA sequence is known, RT-PCR can be used to
map the location of exons and introns in the gene. Another variation is Quantitative PCR (Q-PCR).
It is used to measure the quantity of a PCR product, usually in real time (rt). Q-PCR measures the
amount of DNA, cDNA or RNA. This method has a very high degree of precision partly because it
uses various fluorescent dyes attached to DNA probes to measure the amount of amplified product in
rt. This is why Q-PCR is sometimes referred to as rt-PCR. For more information on PCR techniques,
please visit some of the many on-line methods references listed to the right.
www.clinica lr m.com
| (800) 431-9640