Dr. Charles "Pat" Davis, MD, PhD, is a board certified Emergency Medicine doctor who currently practices as a consultant and staff member for hospitals. He has a PhD in Microbiology (UT at Austin), and the MD (Univ. Texas Medical Branch, Galveston). He is a Clinical Professor (retired) in the Division of Emergency Medicine, UT Health Science Center at San Antonio, and has been the Chief of Emergency Medicine at UT Medical Branch and at UTHSCSA with over 250 publications.
Melissa Conrad Stöppler, MD, is a U.S. board-certified Anatomic Pathologist with subspecialty training in the fields of Experimental and Molecular Pathology. Dr. Stöppler's educational background includes a BA with Highest Distinction from the University of Virginia and an MD from the University of North Carolina. She completed residency training in Anatomic Pathology at Georgetown University followed by subspecialty fellowship training in molecular diagnostics and experimental pathology.
Polymerase chain reaction (PCR) is a technique that is used to amplify trace amounts of DNA (and in some
instances, RNA) located in or on almost any liquid or surface where DNA strands
may be deposited. The key to understanding PCR is to know that every human,
animal, plant, parasite, bacterium, or virus contains genetic material such as
DNA (or RNA) sequences (nucleotide sequences or pieces of DNA or RNA) that are
unique to their species, and to the individual member of that species.
Consequently, if a sample contains segments of DNA or RNA, PCR is a method used
to amplify (make many more identical copies) of these unique sequences so they
can then be used to determine with a very high probability the identity of the
source (a specific person, animal, or pathogenic organism) of the trace DNA or
RNA found in or on almost any sample of material.
PCR amplification is only part of the identifying test, however. Once the
amplification is done (see below), the amplified segments need to be compared to
other nucleotide segments from a known source (for example, a specific person,
animal, or pathogenic organism). This comparison of unique segments is often
done by placing PCR-generated nucleotide sequences next to known nucleotide
sequences from humans, pathogens, or other sources in a separating gel.
Electrical current is run through the gel and the various nucleotide sequences
form bands that resemble a "ladder" according to their electrical charge and
molecular size. This is termed gel electrophoresis. Bands or
"ladder" like steps
that migrate to the same levels in the gel show identity of nucleotide
sequences. This method is one of the most popular ways PCR tests are completed
(See Fig 1).
Figure 1, Bands or "ladder" like steps of PCR produced DNA of
Mycobacterium (courtesy of the CDC)
Figure. Repetitive element (Rep)–PCR (A) and pulsed-field gel electrophoresis (PFGE) (B) patterns of Mycobacterium cosmeticum isolates from 2 patients in Ohio and 1 patient in Venezuela. Rep-PCR was performed by using BOXA1R primer (3), and PFGE was performed with restriction enzyme AseI. Lanes 1, 2, Ohio isolates OH1 and OH2; lanes 3, 4, control strains ATCC BAA-878T and ATCC BAA-879; lane 5, Venezuelan isolate VZ1. DNA size standards are 100-bp (S1) and 48.5-kb marker (S2).
PCR (polymerase chain reaction): PCR (polymerase chain reaction) is a technique in molecular genetics that permits the analysis of any short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. PCR is used to reproduce
(amplify) selected sections of DNA or RNA for analysis. Previously, amplification of DNA involved cloning the segments of interest into vectors for
expression in bacteria, and took
weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is
highly efficient so that untold numbers of copies can be made of the DNA. What
is more, PCR uses the same molecules that nature
uses for copying DNA:
Two "primers", short single-stranded DNA sequences
that are synthesized to correspond to the beginning and ending of the DNA
stretch to be copied;
An enzyme called
polymerase that moves along the segment of DNA, reading its code and
assembling a copy; and
A pile of DNA building blocks that the polymerase needs to make that copy.