Gene Cloning

70 Gene Cloning
(f)
Cycle 3
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Figure 3.21 Polymerase chain reaction. The target DNA, present at the start of the reaction, is
shown in black, and primers in gray; DNA synthesised in each cycle is shown in blue. The region
of the target DNA which is to be amplified is shown in unbroken lines; the flanking regions are
shown as broken black lines. In the first cycle of the PCR reaction template DNA a) is heated to
cause the DNA strands to separate providing single-stranded template for DNA polymerase. The
reaction is cooled to a temperature at which the PCR primers will anneal to the DNA b) and DNA
polymerase is used to synthesize a complementary DNA strand. c). In the first cycle the newly
formed product will begin at each of the primers and extend beyond the sequence complementary
to the other primer, it will terminate when polymerase falls off the template molecule. The
products of this first round of DNA synthesis will be two partially double-stranded molecules of
indeterminate length but beginning at the positions defined by the PCR primers c).
In cycle 2 the temperature of the reaction is again raised to 92°C, the newly formed DNA
duplexes will disassociate providing four template molecules for DNA synthesis d). Two will have
sequences complementary to primer 1 and the other two will have sequences complementary to
primer 2. As the reaction mixture is cooled new primers will anneal and DNA synthesis will begin
again. In cycle 2 the primers will anneal to the genomic template DNA as before and synthesis will
proceed for an indeterminate distance e). However, in cycle 2, primers will also anneal to the
molecules produced in the first round of synthesis. In this case synthesis will begin at the position
where the primer anneals but will stop when the template runs out, this will be the position of
the other primer e). These new molecules will be exactly the same length as the target region,
bounded by the sequences defined by the two primers.
In cycle 3 the process is repeated again f), this time there are eight template molecules. The
two molecules from cycle 2, which are the length of the target region, will give rise to a
complementary strand, also exactly the length of the target region and defined by the positions of
the two primers. These double-stranded molecules are the molecules, which PCR is designed to
make (indicated by a box in panel f)