NaOH treatment to neutralize inhibitors of Taq polymerase.
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 (ssDNA). 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.
Bourke
M T
MT
Connecticut State Police Forensic Science Laboratory, Meriden, USA.
Scherczinger
C A
CA
Ladd
C
C
Lee
H C
HC
D001801:Blood Stains; D004247:DNA / Q000032:analysis; D016172:DNA Fingerprinting / Q000379:methods; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005838:Genotype; D006683:HLA-DQ Antigens / Q000032:analysis / Q000235:genetics; D006801:Humans; D012972:Sodium Hydroxide / Q000494:pharmacology*; D019914:Taq Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D006683:HLA-DQ Antigens; D012972:Sodium Hydroxide; D004247:DNA; D019914:Taq Polymerase