Amelogenin as a target for real time PCR quantitation of forensic templates

PCR is the ubiquitous method of forensic DNA analysis, but prior to amplification, two other processes are crucial to obtaining a satisfactory result: template DNA extraction and template quantitation. Here we will presume the extraction process has been performed, and concentrate on the quantitation step: a focus of recent advances. Real time quantitative PCR (RT-QPCR) is an advantageous alternative to probe hybridisation or fluorescent dye association, which are (respectively) laborious and less accurate procedures. We reviewed the available commercial methods of RT-QPCR and concluded that for our requirements, a more attractive solution was the in-house development and validation of an ultra-rapid, small batch size solution. Our solution is real time detection using the Roche LightCycler 2.0 and the amplification of a 106/112 bp amelogenin amplicon. Melt-curve analysis and back extrapolation to the starting template-dependant crossing point generates results from 32 samples in ∼ 30 min (post PCR assembly) and this approach has advantages in that a positive quantitation result implies that in the SGM Plus™ amplification that follows, at the very least, an amelogenin product should be generated. The use of the amelogenin target also provides an indication of the possibility of PCR product travelling from the separate PCR product room backwards into the clean PCR set-up environment, something that the use of telomerase or β-globin amplicons cannot provide. We have validated the use of our LightCycler-amelogenin based quantitation system and have seen significant improvements in the reliability of quantitation measurements in our forensic laboratories.