What is PCR?
Sometimes called
"molecular photocopying," the polymerase chain reaction (PCR) is a
fast and inexpensive technique used to amplify, or make many copies of, small
segments of DNA. This is necessary because methods used for analyzing DNA
(determining the DNA base pair sequence) require more DNA than may be in a
typical sample. A particularly useful feature of PCR is that it allows the
amplification process to be limited to specifically targeted segments of the
DNA mixture--such as the Y chromosome markers used in genealogical testing.
When your vials of cheek
cells arrive at the lab for testing, they are first mixed with a detergent
which causes the cells to burst open and release their DNA along with other
cell contents. The mixture is then washed with a phosphate containing buffer
(mild salt) solution to dilute cellular debris. With minimal preparation, the
sample is ready and the DNA on the targeted area of a chromosome can be
amplified.
How does PCR work?
PCR is a process based
on the ability of a DNA polymerase enzyme that can synthesize a complementary
strand to a targeted segment of DNA in a test tube mixture of the four DNA base. In
addition, the mixture must also contain two DNA fragments, each about 20 bases
long, called primers, that have
sequences complementary to areas adjacent to each side of the target sequence.
(To do PCR, you need to know the DNA sequence around the region you want to
amplify.) These primers can be constructed in the lab, or purchased from
commercial suppliers. If chosen well, the 20-25 base pair sequence will be
unique in the entire human genome so will match only the place specifically
chosen thus limiting and defining the area to be copied.
The mixture is first
heated to denature (separate) the sides of the double- stranded DNA and then
cooled to allow (1) the primers to find and bind to their complementary
sequences on the separated strands and (2) the polymerase to extend the primers
into new complementary strands. Repeated heating and cooling cycles multiply
the target DNA exponentially, since each new double strand separates to become
two templates for further synthesis. In about 1 hour, 20 PCR cycles can amplify
the target by a millionfold. In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created.
From:
The National Human Genome Research Institute Office
of Science Education and Outreach
The entire cycling
process of PCR is automated and can be completed in just a few hours. It is
directed by a machine called a thermocycler, which is programmed to alter the
temperature of the reaction every few minutes to allow DNA denaturing and
synthesis. To avoid the destruction of needed enzymes in the mixtures by the
high temperatures needed to denature the DNA, enzymes from bacteria that thrive
in hot springs are used for the process.
Why is PCR useful?
Once amplified, PCR
products can be used in many different laboratory procedures; for example, most
mapping techniques in the Human Genome Project rely on PCR.
PCR is also valuable in
a number of newly emerging laboratory and clinical techniques, including DNA
fingerprinting, detection of bacteria or viruses (particularly AIDS), and
diagnosis of genetic disorders and preparing samples for genealogical DNA
testing.
source: contexo.info
nhgri.nih.gov
