Date of Submission

5-3-2026

Document Type

Thesis

Department

Forensic Science

Advisor

David San Pietro

Keywords

PreCRTM, T4 DNA Ligase, Forensic DNA, Degraded DNA, STR Analysis, Peak Height Ratios (PHR)

MeSH

DNA, DNA Ligases, DNA Damage, Tandem Repeat Sequences

LCSH

DNA, DNA ligases, DNA damage

Abstract

This study investigated the comparison between using PreCRTM mix (New England Biolabs) and T4 DNA Ligase (New England Biolabs) to repair degraded forensic whole blood DNA samples for PCR amplification. Degraded DNA presents a significant challenge in forensic STR analysis due to reduced amplification efficiency and allele imbalance. Whole blood samples were extracted, quantified, and artificially degraded using UV exposure. Samples were introduced to multiple treatments, including no damage no repair (NDNR), damage no repair (DNR), PreCRTM repair, T4 DNA ligase treatment, and combined repair approaches. Amplified products were analyzed using capillary electrophoresis, and peak height ratios (PHR) were used to assess allele balance. DNA damage resulted in decreased allele balance and increased variability in PHR (peak height ratio) values, though effects were not consistent across samples. PreCRTM treatment provided the most consistent improvement in allele balance, with several loci showing partial or complete recovery following repair. In contrast, T4 DNA ligase alone demonstrated limited results, and modified ligase conditions produced inconsistent results, including allele dropout in some cases. Combined PreCRTM and T4 treatment resulted in generally balanced PHR values, and showed increased efficiency in some select loci, but did not consistently outperform PreCR alone. These findings suggest that enzymatic repair using PreCRTM may improve STR profiling outcomes in degraded DNA samples, while T4 DNA ligase-based approaches require further optimization. The variability observed across samples emphasizes the need for further research with larger samples sizes.

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