A Modular Real-time PCR Concept for Determining the Quantity and Quality of Human Nuclear and Mitochondrial DNA

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs)--generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets--enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.