Analysis of heat-induced DNA damage during PCR and verification, validation and comparative analysis of two PCR megaplexes

Biological evidence collected at crime scenes are often subjected to forensic deoxyribonucleic acid (DNA) testing. During forensic DNA testing the DNA from the evidence and known samples are extracted, purified, amplified using Polymerase Chain Reaction (PCR), and analyzed using capillary electrophoresis (CE). In order to appropriately compare the profile of the suspect to the evidence, it is essential that interpretation parameters and optimized processing schemes are established. This study endeavors to accomplish this by: first, evaluating whether the PCR temperature cycling is detrimental to the amplification process; and second, by establishing and comparing interpretation parameters for two commonly employed short tandem repeat (STR) megaplexes. To evaluate the effects of temperature cycling on downstream signal, a dynamic systems model was developed, validated, and used to test the effects of temperature on DNA damage and the subsequent fluorescence signal. Though DNA is generally thought to be a stable molecule, heat-induced damage does occur. Specifically, this model assesses the damage to the guanine and cytosine bases during temperature cycling. The model conducts the amplification of a single locus during PCR and generates the peak height observed after capillary electrophoresis. The model was designed to assess not only the effects of heat-induced DNA damage but to also incorporate variability in PCR efficiency. The simulated data indicate that heat-induced DNA damage does not significantly reduce the allelic signal. Also, although changes in PCR efficiency introduce variability in the peak heights at all targets, the peak heights observed with and without heat-induced DNA damage are not significantly different. In fact, the variation in PCR efficiency has a larger effect on the number of amplicons produced than does the heat-induced DNA damage. The second part of this study compares two PCR amplification megaplexes, PowerPlex® Fusion and GlobalFiler®, by evaluating their sensitivities, limits of detection, presence of artifacts, heterozygous peak balance, and ability to amplify minor contributors in DNA mixtures. Analysis of single source samples using weighted least squares regression analysis indicates that PowerPlex® Fusion has greater analytical sensitivities and lower limits of detection at comparable dye channels, and both kits display similar heterozygous balance. However, the GlobalFiler® processing scheme produced fewer artifacts for the various single source samples analyzed, particularly at higher target amounts. Also, analysis of two and three person DNA mixtures indicates that both megaplexes perform equally well when detection of the minor contributor is the criterion