Definition of PCR (polymerase chain reaction)
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.
Three major steps are involved in a PCR. These three steps are repeated for
30 or 40 cycles. The cycles are done on an automated cycler, a device which
rapidly heats and cools the test tubes containing the reaction mixture. Each
step -- denatauration (alteration of structure), annealing (joining), and
extension -- takes place at a different temperature:
- Denaturation: At 94 C (201.2 F), the double-stranded
DNA melts and opens into two pieces of single-stranded DNA.
- Annealing: At medium temperatures, around 54 C (129.2
F), the primers pair up (anneal) with the single-stranded "template" (The
template is the sequence of DNA to be copied.) On the small length of
double-stranded DNA (the joined primer and template), the polymerase attaches
and starts copying the template.
- Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks
complementary to the template are coupled to the primer, making a double
stranded DNA molecule.
With one cycle, a single segment of double-stranded DNA template has thus
been amplified into two separate pieces of double-stranded DNA. These two pieces
are then available for amplification in the next cycle. As the cycles are
repeated, more and more copies are generated and the number of copies of the
template is increased exponentially.
To do PCR, the original DNA that one wishes to copy need not be pure or
abundant. It can be pure but it also can be a minute part of a mixture of
materials. So, PCR has found widespread and innumerable uses -- to diagnose
genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human
evolution, clone the DNA of an Egyptian mummy, etc.. Accordingly, PCR has become
an essential tool for biologists, DNA forensics labs, and many other
laboratories that study genetic material.
PCR was invented by Kary Mullis. At the time he thought up PCR in 1983,
Mullis was working in Emeryville, California for Cetus, one of the first
biotechnology companies. There, he was charged with making short chains of DNA
for other scientists. Mullis has written that he conceived of PCR while cruising
along the Pacific Coast Highway 128 one night on his motorcycle. He was playing
in his mind with a new way of analyzing changes (mutations) in DNA when he
realized that he had instead invented a method of amplifying any DNA region.
Mullis has said that before his motorcycle trip was over, he was already
savoring the prospects of a Nobel Prize. He shared the Nobel Prize in chemistry
with Michael Smith in 1993.
As Mullis has written in the journal Scientific American: "Beginning with a
single molecule of the genetic material DNA, the PCR can generate 100 billion
similar molecules in an afternoon. The reaction is easy to execute. It requires
no more than a test tube, a few simple reagents, and a source of heat."
See also: RT-PCR.
Last Editorial Review: 2/2/2009
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