NaOH Treatment to Neutralize Inhibitors of Taq Polymerase

• Published in: Journal of forensic sciences Vol. 44; no. 5; pp. 1046 - 1050
• Main Authors: Bourke, MT, Scherczinger, CA, Ladd, C, Lee, HC
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-09-1999
• Subjects: Blood; Blood Stains; Deoxyribonucleic acid; DNA; DNA - analysis; DNA Fingerprinting - methods; DNA fingerprints; Enzyme Inhibitors - metabolism; Forensic sciences; Genotype; HLA-DQ Antigens - analysis; HLA-DQ Antigens - genetics; Humans; Sodium Hydroxide - pharmacology; Taq Polymerase - antagonists & inhibitors; Taq Polymerase - metabolism; forensic science; inhibitors; inhibitor removal; polymerase chain reaction; Taq polymerase; DNA typing
• ISSN: 0022-1198; 1556-4029
• DOI: 10.1520/JFS12039J

The introduction of polymerase chain reaction (PCR) into the forensic field has greatly extended the ability to analyze DNA from small or degraded samples. However, one significant problem with PCR analysis is the sensitivity of Taq Polymerase to inhibitors found in many substrates commonly encountered with evidentiary materials. We hypothesize that the most problematic of these compounds intercalate into double stranded DNA (dsDNA) and have significantly less affinity for single stranded DNA (ss-DNA). This study presents a comprehensive analysis of a novel method for the neutralization of Taq inhibitors by denaturation and washing with NaOH in Microcon-100 filtration units. The data show that DNA recovered following NaOH repurification routinely amplifies when other inhibitor neutralization techniques are unsuccessful. Genetic profiles have been obtained with both AmpliType PM + DQA1 and D1S80 systems. However, the NaOH protocol is not advised when the quantity of DNA is limited since the treatment results in significant loss of DNA.