7 Assessment of STR profiles
DNA profiles generated from casework samples require some experience to interpret. Guidelines have evolved to assist with the interpretation of STR profiles, ensuring that the results are robust and consistent; this is especially important when dealing with samples that contain very small amounts of DNA, degraded DNA or mixtures of profiles that come from two or more individuals – all situations that complicate interpretation. This chapter explores a number of artefacts that can occur in DNA profile. Some casework scenarios that can lead to complex profiles are also considered.
During the amplification of an STR allele it is normal to generate a stutter peak, that is one repeat unit smaller or larger that the true allele; smaller alleles are formed in the majority of cases . Stutter peaks are formed by strand slippage during the extension of the nascent DNA strand during PCR amplification (Figure 7.1) [2, 3]. Even in good quality profiles there will be some stutter peaks; these are recognizable and do not interfere with the interpretation of the profile. Threshold limits are normally used to aid in the identification and interpretation of stutter peaks, so, for example, while the degree of stutter varies between loci, they are typically less than 15% of the main peak [4, 5] – understanding stutter peaks is especially important when interpreting mixtures. Different STR loci have varying tendencies to stutter. This is dependent on the structure of the core repeats: shorter di- and trinucleotide repeats are more prone to stutter than are tetra- and pentanucleotide repeats and this is one of the reasons that all the autosomal STRs that have been adopted by the forensic community have tetra and pentanucleotide core repeats (Figure 7.2). STRs with simple core repeats tend to have higher stutter rates than compound and complex repeats.
Split peaks (+/− A)
The Taq polymerase that is used to drive the polymerase chain reaction adds nucleotides to the newly synthesized DNA molecule in a template-dependent manner. However, it
Figure 7.1 During PCR, slippage between the template and the nascent DNA strands leads to the copied strand containing one repeat less than the template strand also has an activity, called terminal transferase, whereby it adds a nucleotide to the end of the amplified molecule which is non-template-dependent . Approximately 85% of the time an adenine residue is added (Figure 7.3). It is important that the vast majority of PCR products have the non-template nu- cleotide added, otherwise a split peak is observed in the DNA profile (Figure 7.4). Split peaks are usually caused either by the sub-optimal activity of the Taq 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 65-72 ◦C for between 45-60 minutes, allowing the Taq 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 one base pair; the THO1 9.3 allele for example could be confused with the THO1 allele 10. In most cases a profile with a high degree
Figure 7.2 Stutter peaks are formed during slippage of the Taq polymerase during replication of the template strand. The slippage results in amplification products one repeat unit shorter than the template. The stutter peaks are normally less than 15% of the true amplification product. Panel (a) shows a dinucleotide repeat, which is prone to high levels of slippage, the stutter peaks are indicated by the arrow and their size relative to the main peak is shown (based on peak area). Panel (b) is a tetranucleotide repeat, which displays lower levels of stutter