Figure 7.3 The Taq polymerase adds a nucleotide to the 3′ end of the newly synthesized strand. The non-template addition is usually an adenine and results in a PCR product that is one base pair longer than the template (N + 1). The arrowed lines represent the forward and reverse primers of split peaks would have to be re-analysed to minimize the possibility of incorrect interpretation.
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 GeneMapperTM 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 sized 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).
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 panel (a) showing an example where the vast majority of PCR product has the non-template addition through to panel (c), where only 50% of the PCR product has the non-template addition
Figure 7.5 If the reaction is overloaded with DNA, (a) the peaks are still present but artefacts such as pull-ups and split peaks are more pronounced. When the template is within the optimal range (b and c) the peaks are well balanced and easy to interpret. When the PCR does not have enough template to amplify (d), then locus and allelic drop-out can occur (see plate section for full-colour version of this figure)