Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples

Abstract

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10–100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1–1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

“…by mutations [11]. In the current study, sample type had a notable impact on the performance of the two tested alternative DNA polymerases.…”

“…However, AmpliTaq Gold might not be the optimal choice for samples containing PCR inhibitors. In several studies, AmpliTaq Gold has shown lower tolerance to PCR-inhibitory substances compared with other DNA polymerases [7][8][9][10][11]. We have previously shown that short tandem repeat (STR) DNA analysis of complex crime scene samples can be improved by replacing AmpliTaq Gold with either of the alternative DNA polymerase-buffer systems Bio-X-Act Short (Bioline, London, UK), ExTaq Hot Start (TaKaRa Bio, Shiga, Japan), or PicoMaxx High Fidelity (Stratagene, La Jolla, CA, USA) [6].…”

Forensic Genetics and Genotyping

“…proteases from food particles, bacterial cells) (14) than in blood (e.g. heme, hemoglobin, lactoferin, immunoglobin G) (15,16,17). Saliva transports exfoliated epithelial cells from buccal mucosa which contains DNA and the mean number of epithelial cells per 1 mL of saliva is about 4.3 × 10 5 , which makes it a suitable source of genomic DNA (8,11,18).…”

Towards autonomous lab-on-a-chip devices for cell phone biosensing

“…Instead of solid-phase extraction, in the selected example, PCR is directly performed by inactivating the inhibitors in the sample. In addition, an inhibitor-resistant Taq mutant (Kermekchiev et al, 2009) of the Taq polymerase enzyme is used, which minimizes the incubation period. The device was tested with the identification of the 108-bp segment of the DYZ1 region within the human genome, which has application in sex determination (Jobling et al, 1997).…”