Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples.
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.
Kermekchiev
Milko B
MB
DNA Polymerase Technology Inc., Saint Louis, MO 63104, USA. milko@klentaq.com
Kirilova
Lyubka I
LI
Vail
Erika E
EE
Barnes
Wayne M
WM
D052160:Benzothiazoles; D004247:DNA / Q000032:analysis* / Q000097:blood; D003959:Diamines; D004791:Enzyme Inhibitors / Q000097:blood / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D009154:Mutation; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D017384:Sequence Deletion; D012988:Soil Microbiology; D019914:Taq Polymerase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
D052160:Benzothiazoles; D003959:Diamines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA; D019914:Taq Polymerase