Reliable nuclear and mitochondrial DNA quantification for low copy number and degraded forensic samples

Abstract

DNA quantification is a prerequisite for both low copy number (LCN) forensic analysis and ancient DNA (aDNA) studies. Moreover, if nuclear quantification is focused on the amelogenin locus, it also allows sex determination. Some of the problems of these techniques are allelic drop-out phenomenon in amelogenin locus and mitochondrial DNA (mtDNA) quantification biases, due to human intraspecific variation affecting the annealing of primers and/or probes.The method presented here combines two multiplex TaqMan (R) real-time PCR (qPCR) for nuclear and mtDNA quantification in degraded or limited samples. Nuclear DNA detection is based on the independent amplification of X and Y chromosome specific fragments in the amelogenin locus and an internal PCR control (IPC) to recognize inhibition problems. The small length of the fragments (71 bp) favors the quantification of severely degraded DNA, whereas the use of two distinct primer sets for X and Y chromosome amplification is directed to reduce allelic drop-out in LCN analysis. MtDNA quantification is based on the amplification of three PCR fragments located in the mtDNA 16S region. Two of them are amplified with human specific conservative primers and probes, which allows a world-wide application of this technique. Moreover, their length difference (167 and 314 bp respectively), provides information about the DNA degradation level. In order to also recognize non-human DNA an interspecific mtDNA fragment (187 bp) was also designed.