Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains

Abstract

Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5' defined sequence and alter the number of bases on the 3' end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors.

Generating DNA profile from low copy number DNA: Strategies and associated risks

“…As these kits contain functionally efficient primers that can bind to small fragment of DNA, it has been proposed that these can be good candidates for amplification of LCN obtained from various forensic evidence (Butler, Shen, & McCord, 2003;Mulero et al, 2008). With the use of modified nucleotides in place of standard nucleotides, increase in the efficiency of polymerase chain reaction and proper functioning of these primers has proven effective in amplifying low copy number of DNA samples (Ambers et al, 2016;Strom & Rechitsky, 1998).…”

Single-nucleotide polymorphism analyses on ABCC11, EDAR, FGFR2, and ABO genotypes of mummified people of the Joseon Dynasty, South Korea

“…Lalueza-Fox et al (2008) found a humanspecific diagnostic deletion for blood group O (O01 haplotype) in two Neanderthal skeletons unearthed at the El Sidron site. Ambers et al (2016) introduced a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)-based method for ABO genotyping of degraded and low-copy templates. As for ancient human samples obtained from archaeological sites in South Korea, Kim et al (2011) utilized a multiplex SBE method to determine the blood types of two pre-modern Korean mummies (AO01 and AB).…”

“…However, insufficient reports are available to determine the potential of this methodology for the analysis of ancient human specimens. In fact, with respect to the SNPs in the ABCC11, EDAR, FGFR2, and ABO genes, only a very limited number of papers on ancient specimens obtained at archaeological sites are currently available (Lalueza-Fox et al, 2008;Kim et al, 2011;Keller et al, 2012;Ambers et al, 2016).…”

Validation of Reduced-volume Reaction in the PowerQuant® System for human DNA Quantification

“…Quantitative PCR (qPCR) has played an essential role in DNA analysis since its first introduction in the forensic field (Westring et al, 2007;Ambers et al, 2016;Holt et al, 2016). It is a sensitive method for DNA quantification and…”