Characterization of the effects associated with amplification of degraded DNA using traditional and mini-STRs
Dunn, Jonathon Stephen
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Many samples submitted for DNA analysis from crime scenes are often degraded or contain DNA mixtures from two or more individuals. Degradation of DNA in these samples presents a challenge as larger molecular weight STR markers are inefficiently amplified, if they are amplified at all, potentially resulting in a significant loss of information. Furthermore, deducing individual donors from DNA mixtures is difficult in the best of times. However, when one or more of the contributors to the mixture exhibit degradation, analysis of the mixtures becomes even more challenging. Recent advancements in the amplification of mini-STRs has led to an increased ability to gain information from degraded or inhibited samples that may otherwise be lost using traditional STR amplification. The commercially available Mini Filer™ kit was developed using the eight largest loci from the AmpFℓSTR® ldentifiler® kit plus the Amelogenin locus by redesigning the primers such that shorter PCR products are produced, relative to previous STR kits. Due to this redesign, the MiniFiler™ kit was shown to increase the likelihood of obtaining information otherwise lost in these eight loci. The goal of this research is to compare the genotyping success obtained using mini- and traditional-STR amplification in both degraded and non-degraded single-source samples and mixtures. The objectives include 1) comparing peak height ratios of heterozygous markers and drop-out rates of the single source samples between the kits 2) observing whether these ratios are maintained in mixtures and 3) to examine if the DNA within the mixtures amplify independently. DNA from one male and one female were degraded using DNAse 1. Both the male and female samples were run as single-source samples in both the degraded and non-degraded conditions in quadruplicate using both kits with targets ranging from 0.125-2 ng. Mixtures ofthe two samples containing either degraded or non-degraded DNA were run using both kits with varying female to male ratios. Finally, mixtures using the same female to male were run using the degraded female sample and the non-degraded male sample. All mixture samples were also run in quadruplicate. Results showed that the mini-STR amplification did not outperform traditional-STR amplification in non-degraded DNA samples. As a result, it is recommended that traditional PCR methods be used with non-degraded samples. In contrast, mini-STR amplifications did prove to be successful in recovery of allelic dropout in degraded samples. Therefore, for exhaustive biological evidence, a priori knowledge of sample conditions would be beneficial as only samples that are expected to contain degraded DNA should be amplified with mini-STRs. If the quantity and quality of DNA is deemed sufficient, then it is recommended that traditionai-STR methods be utilized and mini-STR amplification follow.
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