The PCR (Figure 9-3) involves three temperature incubations or
steps that are repeated 20-50 times. One repetition of three steps is called
a cycle. In the first step, called denaturation, the two strands of the target
DNA molecule are separated (denatured) by heating the DNA to 94°C
to break the hydrogen bonds between bases, yielding two separate
strands. In the second step, called annealing, two primers hybridize to
complementary sequences in the single strands. The primers are short
(20-30 bases in length), synthetic stretches of single-stranded DNA .
They are selected so that one primer is complementary to one end of the
gene of interest on one strand, while the second primer is complementary
to the opposite end on the other strand. The primers form hydrogen bonds
with (anneal to) their complementary sequences, forming stable, doublestranded
molecules. Annealing temperatures range between 37 and 60°C.
During the third step, extension, or elongation, the primers are extended
by a thermostable DNA polymerase at 72°C.
|Figure 9-3 Schematic representation of a typical PCR assay.
To study the effects of mutations on gene expression, researchers
have developed a technique known as site-directed mutagenesis, which
introduces point mutations at specific sites. One of the most commonly
used strategies takes advantage of primer-directed amplification of DNA
to introduce mutations. One of the primers is designed with a sequence
complementary to the region in the target DNA , but with the desired substitution,
insertion, or deletion. The mutagenic sequence within the
primer must be either at the 5' end of the primer or internal to the primer,
but never at the 3' end of the mutagenic primer. The 3' end of the mutagenic
primer (at least 6-10 bp long) must be totally complementary to
the target DNA to permit full annealing of the primer to its target and allow
the polymerase to extend the primer. The PCR is carried out initally
(first 5-10 cycles) under low stringency conditions, to allow the mismatch
to occur. Once a few mutagenized templates are produced during
the PCR, these will serve as targets and will be fully complementary to
the primer. The end products will contain the mutation at the desired site.
The steps of PCR: