Biology of Disease

The PCR consists of a series of cycles, each of which consists of three identical
steps (Figure 3.28). First, the double-stranded DNA has to be heated to 90–
96°C to break the hydrogen bonds that connect the two strands and allow
them to separate. This denaturation is called melting. Prior to the first cycle,
the DNA is often heated for an extended time, called a ‘hot start’, of up to 5
to 10 min to ensure that the template DNA and the primers are fully melted.
In subsequent cycles, 30 s to 3 min at 94 to 96°C is normally sufficient for
melting. Placing a heated lid on top of the reaction tubes or a layer of oil
on the surfaces of the reaction mixtures prevents evaporation. The second
step is primer annealing at temperatures of 50 to 65°C for 30 to 60 s, during
which the primers bind to their complementary bases on the now single-
stranded DNA templates. The primers must be present in excess of the target
DNA otherwise its strands will simply rejoin. The design of the length of the
primers requires careful consideration. Primer melting temperature, which
must not be confused with that of the template DNA itself, increases with
the length of the primer. The optimum length for a primer is generally 20 to
40 nucleotides that have melting temperatures of 60 to 75°C, although this
depends upon their G/C content. If the primers are too short they will anneal
at random positions on the relatively long template and result in nonspecific
amplification. However, too long a primer and the melting temperature would
be so high, that is above 80°C, that the DNA polymerase would have reduced

INVESTIGATING INFECTIOUS DISEASES

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Figure 3.28 Outline of the steps associated with the cycles of the polymerase chain reaction.
See text for details.

Melting Annealing Extension Melting

30s–3min 30s–1min 45s–2min 30s–3min

Duration

Temperature / °C

94–96

60–75

50–65

5'

5'

3'

3'

5' 3'

3' 5'

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DNA

Primer strands