Biology Reference
In-Depth Information
(a) (b) (c)
Figure 7.4
Split peaks are seen in profiles when the non-template addition does not occur with all
of the PCR products. The three examples show decreasing amounts of non-template addition with
(a) showing an example where the vast majority of PCR product has the non-template addition,
(b) with around 25% non-template addition through to (c) where only 50% of the PCR product has
the non-template addition
It is important that the vast majority of PCR products have the non-template
nucleotide added, otherwise a split peak is observed in the DNA profile (Figure 7.4).
Split peaks are usually caused either by the suboptimal activity of the
Ta q
polymerase
or by too much template DNA in the PCR.
In order to minimize the formation of split peaks in a profile, at the end of the
cycling stage of the PCR, the reaction is incubated at 65
◦
C-72
◦
C for between 45
and 60 minutes, allowing the
Ta q
polymerase to complete the non-template addition
of all the PCR products.
The interpretation of profiles with split peaks is possible because the peak with
the nucleotide added is taken as being the correct peak. Problems can occur when
alleles are present that differ by only 1 bp; the TH01 9.3 allele, for example, could
be confused with the TH01 allele 10. In most cases, a profile with a high degree
of split peaks would have to be re-analysed to minimize the possibility of incorrect
interpretation.
Pull-up
In Chapter 6 the matrix file was introduced; this file contains information about the
levels of spectral overlap that exist with the dyes that have been used to label the
PCR products. This information is used by the Genescan
and GeneMapper
TM
ID
software to produce peaks that are made up of one colour. If the matrix file is not of
good quality then this correction is not perfect and the peaks in the resulting profile
are composed of more than one colour; this phenomenon is called pull-up. Pull-ups
are easy to recognize as a smaller product will appear at exactly the same size as
the real STR allele. Pull-up can also occur when there has been over-amplification,
even if the matrix file is of good quality (Figure 7.5a).
