title: Pressure cycling technology (PCT) reduces effects of inhibitors of the PCR.
abstract: A common problem in the analysis of forensic human DNA evidence, or for that matter any nucleic acid analysis, is the presence of contaminants or inhibitors. Contaminants may copurify with the DNA, inhibiting downstream PCR or they may present samples effectively as containing fewer templates than exist in the PCR, even when the actual amount of DNA is adequate. Typically, these challenged samples exhibit allele imbalance, allele dropout, and sequence-specific inhibition, leading to interpretational difficulties. Lessening the effects of inhibitors may increase the effective yield of challenged low template copy samples. High pressure may alter some inhibitors and render them less effective at reducing the yield of PCR products. In an attempt to enhance the amplicon yield of inhibited DNA samples, pressure cycling technology was applied to DNA exposed to various concentrations of hematin (0, 1.25, 2.5, 5, and 7 μM) and humic acid (0, 1.25, 2.5, 5, and 7 ng/μL). The effect of high pressure on the inhibitors, and subsequently the PCR process, was assessed by measuring DNA quantity by quantitative PCR and evaluating short tandem repeat typing results. The results support that pressure cycling technology reduces inhibitory effects and thus, in effect, enhances yield of contaminated amplified products of both hematin and humic acid contaminate samples. Based on the results obtained in this study, this method can improve the ability to type challenged or inhibited DNA samples.
authors.affiliation: Institute of Applied Genetics, Department of Forensic and Investigative Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA. pamela.marshall@unthsc.edu
authors.forename: Pamela L
authors.initials: PL
authors.lastname: Marshall
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: King
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Lawrence
authors.forename: Alexander
authors.initials: A
authors.lastname: Lazarev
authors.forename: Vera S
authors.initials: VS
authors.lastname: Gross
authors.forename: Bruce
authors.initials: B
authors.lastname: Budowle
mesh_terms: D004247:DNA / Q000032:analysis* / Q000187:drug effects*; D059018:DNA Contamination; D016172:DNA Fingerprinting; D053803:Forensic Genetics / Q000379:methods*; D006427:Hemin; D006801:Humans; D006812:Humic Substances; D018895:Microsatellite Repeats; D011312:Pressure; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D006812:Humic Substances; D006427:Hemin; D004247:DNA