Biology Reference
In-Depth Information
The normal range of cycles for a PCR is between 28 and 32. In extreme cases,
where the amount of target DNA is very low the cycle number can be increased to
up to 34 cycles. It has been demonstrated that going above this cycle number does
not increase the likelihood of obtaining a profile but does increase the probability
of artefacts forming during the PCR [18]. Using 34 cycles is known as low copy
number (LCN) PCR
1
and it is sparingly employed, as extreme precautions have to be
taken to reduce the chance of contamination; the more cycles the higher the chance
of detecting contaminating DNA. The interpretation of the profiles generated using
a high cycle number also become more complex.
Following the cycling phase the reaction is incubated between 60
◦
C and 72
◦
C
for up to 1 hour. In addition to the template-dependent synthesis of DNA, the
Ta q
polymerase also adds an additional residue to the 3
end of extended DNA molecule,
this is non-template dependent [31]; the incubation at the end of the reaction is to
ensure that the non-template addition is complete. The conditions of a typical PCR
are shown below in Figure 5.3.
The PCR requires tightly controlled thermal conditions and these are achieved
by using a thermocycler. This consists of a conducting metal block that contains
heating and cooling elements with wells that accommodate the plastic reaction tubes.
The temperature of the PCR block is controlled by a small microprocessor. Most
thermocyclers also contain a lid, which is heated to over 100
◦
C; this prevents the
reaction evaporating and condensing on the cooler lid and thereby maintains the
reaction volume, thus keeping the concentration of the reaction components stable
throughout the PCR.
After amplification the results of a PCR can be visualized on an agarose gel. In a
reaction that amplifies only one locus a single band should be detected
2
(Figure 5.4).
PCR inhibition
When analysing forensic samples a problem that can be encountered is inhibition of
the PCR [32]. DNA extraction methods do not produce pure DNA; some chemicals
1 Hold
3 Tmp 28 cycles
2 Holds
94.0
94.0
72.0
59.0
60.0
11:00
1:00
1:00
65:00
1:00
4.0
∞
Method: SGM+
_
Figure 5.3
A typical PCR programme involves three phases: a pre-incubation at 94
◦
C, which
activates the AmpliTaq Gold
polymerase; the cycling phase; and a terminal incubation that
maximizes the non-template addition at the end of the amplification. The programme shown is for
amplification using the SGM Plus
(see Chapter 6)
1
Note: The term low copy number PCR is now commonly referred to as low template number PCR.
2
Note: An exception would be when the target locus contains alleles of varying length, such as VNTRs, AMP-FLPs and
STRs.
