Gene Amplification: Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR) is a procedure that borrows a cell's machinery for DNA replication, allowing researchers to make many copies of a gene of interest. Starting materials are:
two
types
of
short
DNA
pieces
known
to
bracket
the
gene
of
interest,
called
primers.
a
large
supply
of
DNA
bases.
the
enzymes
that
replicate
DNA.
A simple test procedure rapidly builds up copies of the gene. Here's how it works:
In the first step of PCR, heat is used to separate the two strands of the target DNA-such as bacterial DNA in a body fluid sample from a person who has symptoms of an infection. Next, the temperature is lowered and the two short DNA primers are added. The primers bind by complementary base pairing to the separated target strands. In the third step, DNA polymerase and bases are added. The DNA polymerase ads bases to the primers and builds a sequence complementary to the target sequence. The newly synthesized strands then act as templates in the next round of replication, which is immediately initiated by raising the temperature. All of this is done in an automated device called a thermal cycler that controls the key temperature changes.
The pieces of DNA accumulate geometrically. The number of amplified equals the number of temperature cycles. After just twenty cycles, one million copies of the original sequence are in the test tube.
PCRs greatest strength is that it works on crude samples of rare and short DNA sequences, such as a bit of brain tissue on the bumper of a car, which in one criminal case led to identification of a missing person. PCR's greatest weakness, ironically, is its exquisite sensitivity A blood sample submitted for diagnosis of an infection contaminated by leftover DNA from a previous run, or a stray eyelash dropped form the person running the reaction, can yield a false positive result. The technique is also limited in that a user must know the sequence to be amplified and that mutations can sometimes occur in the amplified DNA that are not present in the source DNA.
