Chapter 19 - Low Copy Number Stochastic Results

Abstract

Commercial multiplex short tandem repeat (STR) DNA typing kits used in crime laboratories work optimally under a narrow range of conditions. Even if prior DNA quantitation steps detect no DNA, amplifiable DNA may be present. Low copy number (LCN) is defined as less than 100 picograms of DNA. This is the stochastic threshold beyond which polymerase chain reaction (PCR) amplification is not reliable. LCN DNA may also be referred to as touch DNA, trace DNA and low-level DNA.In this experiment, a polymerase chain reaction (PCR) will be performed to evaluate the production of the STR profile for low copy number DNA samples serially diluted from a known stock sample of a standard DNA template. Three sets of PCR primers will be used to probe the D7S820, D5S818, and Penta D loci and produce heterozygous amplicons. Real-time (or standard) PCR will be conducted using standard K562 DNA, the three sets of PowerPlex16 PCR primers, and Bio-Rad’s iQ SYBR Green Supermix employing SYBR Green I. The reaction mix will be pipetted to a 96-well plate and amplifed using a Bio-Rad iQ5 or similar instrument. Evaluation of the melting temperature using a melt curve and first derivative plot will be used to qualitatively determine the production of the correct amplicons for the three size ranges. At the completion of the PCR experiment, the plate may be frozen until the next laboratory session in which 2% agarose gels of the PCR products will be run to confirm the length of the amplicon with an appropriate low molecular weight DNA ladder to detect heterozygous peak imbalance resulting in only one detectable allele from a heterozygous pair or total allele dropout. The observed alleles will be assigned and the results will be tabulated for each locus and dilution.