TY - JOUR
T1 - Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples
AU - Kermekchiev, Milko B.
AU - Kirilova, Lyubka I.
AU - Vail, Erika E.
AU - Barnes, Wayne M.
N1 - Funding Information:
This work was supported by the National Institutes of Health (SBIR Grant 2R44GM07340102 to M.K.); and the US Department of Agriculture (SBIR Grant 2006-03071 to M.K.). Funding for open access charge: National Institutes of Health (SBIR Grant 2R44GM07340102 to M.K.).
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=63249099357&partnerID=8YFLogxK
U2 - 10.1093/nar/gkn1055
DO - 10.1093/nar/gkn1055
M3 - Article
C2 - 19208643
AN - SCOPUS:63249099357
SN - 0305-1048
VL - 37
JO - Nucleic acids research
JF - Nucleic acids research
IS - 5
M1 - e40
ER -