Commercial STR kits have been optimized to amplify small amounts of template DNA, commonly between 0.5 and 2.5 ng, which represents approximately 166 and 833 copies of the haploid human genome. It is not always possible to add the optimum amount of DNA to a PCR when the sample size is limited.
Overloading the PCR can also lead to a profile that is difficult to interpret. If the CCD camera is saturated then the peak height/area is no longer a good indicator of the amount of product and this can lead to problems in assessing peak balance and can make the interpretation of mixtures difficult. Overloaded profiles also tend to have a noisy baseline, increased levels of stuttering, split peaks and pull-ups (Figure 7.6).
Low copy number DNA
At many crime scenes it may be possible to infer surfaces with which the perpetrator has had physical contact, for example the handle of a gun, a knife, a ligature, a door handle or a steering wheel. These areas can be swabbed to collect any epithelial cells that have been shed during the contact [7-10]. The amounts of DNA extracted can be extremely low but in some circumstances it is possible to get a full DNA profile from less than 100 pg of template DNA: the normal range of template DNA is between 500 and 2500 pg (2.5 ng). To analyse such small quantities of DNA the number of amplification cycles is increased to 34. The standard number of cycles in the amplification using commercial kits is between 28 – 32 cycles. Empirical studies have shown that above 34 cycles the
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 (see plate section for full-colour version of this figure)
amount of artefacts that are detected outweigh the benefit of higher levels of artefact .ExtremecarehastobetakenwheninterpretingtheLCNprofiles[11,12].Anumber 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 [12-14]. 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. To minimize the possibility of this occurring the PCR must be repeated at least two times and only alleles that appear consistently 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 and are not amplified in the duplicate or triplicate reactions but can still confuse the interpretation of the profile. Locus drop-out, particularly of the larger STR loci, can also occur; this reduces the amount of information from the profile but does not confuse the interpretation. At present, there is no clear consensus in the scientific community about the use of LCN PCR .
Figure 7.7 Three separate PCR analyses of a DNA extract can lead to different results. When dealing with very low template numbers, allelic dropout is relatively common and the true genotype can only be ascertained through multiple amplifications – and even then the results can be contentious