A quantitative PCR assay for the assessment of DNA degradation in forensic samples

Abstract 

A multiplex quantitative PCR assay has been designed to amplify target sequences of different length, which allows for the assessment of DNA degradation in samples of forensic interest. The targets were chosen to provide quantification and fragment length information relevant to the STR amplification targets commonly used for forensic genotyping. The longer target (nuTH01, 170–190bp) spans the TH01 STR locus. Although not one of the longest loci used for STR genotyping, it was chosen as a good compromise given the target length limitations on qPCR efficiency with TaqMan^® detection. The shorter target (nuCSF, 67bp) was designed in the upstream flanking region of the CSF1PO STR locus. In addition to these human nuclear targets, the assay includes an internal PCR control target sequence to allow for an assessment of PCR inhibition. The assay was rigorously tested on samples with varying amounts of degradation, and the ratio of nuCSF:nuTH01 quantifications was shown to provide a good estimation of the degree of degradation present in a sample. This estimate, along with the internal control for PCR inhibition, provides a valuable tool for post-extraction sample assessment.

Keywords: Forensic sciences, DNA quantitation, Quantitative polymerase chain reaction, Degraded DNA, TH01, CSF