Campbell, Chapter 20; 6
Ed. pages 382-386, 393
Ed. 391-398. In 7
Ed. only; add page 399 to Genomes lecture reading
The polymerase chain reaction (PCR) is used to amplify, or make many copies of, a given
DNA sequence, in vitro (that is, in a test tube, without cells). This contrasts with the gene cloning
we discussed last time, which is done by introducing genes into a plasmid, putting the recombinant
plasmid in bacteria, and letting bacteria replicate the plasmid. In addition to the fact that we don’t
need bacteria, the advantages of PCR are the following: it’s fast, it needs very little starting
material, and it’s not limited by a need for restriction enzyme recognition sites. Any piece of DNA
can be amplified.
For PCR, we need a DNA template, a
special heat-stable DNA polymerase (that
can withstand the heat required to denature
the DNA strands), dNTPs, and TWO
primers that are complementary to short
sequences on OPPOSITE STRANDS of the
targeted sequence. The 5’ end of each
primer marks one end of the targeted
sequence (see Fig. 1). The primers are
included in huge excess over the template.
The method is to repeat the following 3-step
cycle 25 or 30 times (See Campbell, Fig.
20.7, both Eds). Step 1; heat the DNA to denature (i.e., to separate the 2 strands).
Step 2; cool to allow complementary base pairing of the template and the primers.
(Primers bind template more often that template strands bind each other, because
they’re present in excess). Step 3; extend, allowing DNA synthesis to occur from the primers, using
the template DNA as a guide. During the first cycle, each primer binds a strand of the template, and
is extended into a new long strand. After awhile, we stop the extension reaction and start Cycle 2,
by heating again. When we do this, the new strand and the template strand come apart from each
other. We now cool to let new primers bind again. At this point, each primer has 2 choices. It can
either bind to the complementary original template strand. If it does this, during extension, it will
make another strand, just as in Cycle 1. However, instead, the primer might bind to the new strand
that was made in Cycle 1 starting with the OTHER primer. (This new strand has the same sequence
as the template strand that our primer recognizes). Note that this new strand is not very long. For
this reason, if a primer binds to it, and DNA polymerase uses it as a template during the extension
reaction of Cycle 2, it will soon reach the end. This strand (made in Cycle 2) will be short – only as
long as the distance between the 2 primers. In other words, it’s the desired fragment!
Each new strand that’s made in the PCR reaction can serve as a template in the next cycle.