Development and use of triplex real-time PCR assay for human DNA quantification in forensic samples

Abstract

It is usual for the human DNA samples encountered in forensic casework to be variably degraded, thus signifi cantly affecting downstream analysis. Furthermore these samples are commonly contaminated with micro-organisms, so that total DNA extraction from the samples will contain variable amounts of non-human DNA, thus diffi culting the quantifi cation of the human DNA component by standard UV absorbance at 260 nm. It has become almost routine to quantify the human DNA component of forensic DNA extractions by using realtime PCR (qPCR) and commercially sold kits are presently available for this. We describe the development and use of a single-tube triplex qPCR assay for quantifying human DNA in extracted forensic samples prior to amplifi cation of STR loci. Two nuclear loci are targeted, one being autosomal and the other situated on the Y chromosome. The third target is a synthetic oligonucleotide which is present during amplifi cation and which acts as an internal control for amplifi cation inhibition. Three different common fl uorophores are used and the chemistry is based on a quencher molecule linked to an antiprimer which suppresses fl uorescence until the generation of suffi cient amplicon during amplifi cation overcomes the quenching. The amplicon sizes are approximately 80 bp. Signal generation is monitored in a qPCR instrument in the presence of a passive reference dye. We demonstrate that the qPCR triplex in question is an important tool for our forensic analyses, because it allows us to obtain information concerning the quantity and quality of the human DNA in our extracted samples, prior to using the costly human identifi cation reagents, such as in the analysis of STRs, SNPs and DIPs.