A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA

Niederstätter,H.; Köchl,S.; Grubwieser,P.; Pavlic,M.; Steinlechner,M.; Parson,W.
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 246bp; mtDNA: 102, 143, 283, and 404bp) 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
Forensic Sci Int Genet 2007 1(1):29-34
Tags: amplification; DNA; DNA Fragmentation; genotyping; human; in vitro; mitochondrial DNA; mtDNA; Mutagenesis; PCR; PCR PRODUCTS; PRIMERS; PRODUCTS; rtPCR; system
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