Biology Reference
In-Depth Information
Split peaks
Stutter
Noisy baseline
Figure 7.6
A heavily overloaded profile. All the peaks shown have a flat top, indicating that they
are off-scale, the baseline is very noisy, several split peaks are evident and the peaks are very
broad which can lead to sizing problems. There are also some pronounced stutter peaks
number of amplification cycles is increased to 34, termed low copy number (LCN)
when introduced by the Forensic Science Service, UK. The standard number of cycles
in the amplification using commercial kits is between 28 and 32 cycles. Empirical
studies have shown that above 34 cycles the amount of artefacts that are detected
outweigh the benefit of higher levels of artefact [10]. Extreme care has to be taken
when interpreting LCN profiles (low template number) [10, 11]. A number of features
can be seen when amplifying low amounts of template DNA. These are allele drop-
out and drop-in; severe peak imbalance; locus drop-out (Figure 7.5d); and increased
stutter [11 - 13]. Allele drop-out occurs when through chance events one allele in a
heterozygous locus is preferentially amplified; this can give the false impression that
the profile at a particular locus is homozygous. PCR must be repeated at least twice
to minimize the possibility of this occurring and only alleles that appear in two or
more amplifications can be called (Figure 7.7).
This phenomenon also leads to a peak imbalance that is much higher than when
using higher amounts of template DNA. Allele drop-in is also a common phenomenon
when amplifying low amounts of template DNA. The drop-in alleles are spurious
amplification products from unknown DNA, such as cells from operators and on
plasticware. As allele drop-in is a random event; it is unlikely that the same allele
will be amplified in both samples of a duplicate or triplicate reactions, but they can
still confuse the interpretation of the profile. Locus drop-out, particularly of the larger
D21S11
D21S11
D21S11
Drop-out
Figure 7.7
Three separate PCR analyses of a DNA extract can lead to different results. When dealing
with very low template numbers allelic drop-out is relatively common and the true genotype can
only be ascertained through multiple amplifications; even then the results can be contentious
