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

Kermekchiev MB, Kirilova LI, Vail EE, Barnes WM - Nucleic Acids Res. (2009)

Bottom Line: 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.Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t.Taq polymerase as compared to the mutant enzymes.

Affiliation: DNA Polymerase Technology Inc., Saint Louis, MO 63104, USA. milko@klentaq.com

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.

Effect of the extension time on DNA amplification from blood (endogenous targets) or crude soil extract. Two targets of the human CCR5 and DNMT genes, 1.1 kb and 0.5 kb, respectively, were amplified in duplex PCR directly from 1.25% to 10% human blood with KlenTaq 10, Taq 22, or w.t. Taq (lanes 1–4, A). Control reactions (lanes C) contained 10 ng DNA and no blood. In (B), the same targets were amplified with the three enzymes from 10 ng human DNA, in the presence of 0.5–20% crude soil extract (lanes 1–5) (Note the shift in the range of the soil extract used with the w.t. enzyme). Control reactions (lanes C) contained no soil extract. Two identical samples of each reaction were amplified with 4 min or 2 min extension time in a no ramp-time PCR cycle. Lanes M, DNA standards ladder. The amplified products were analyzed in a 2% agarose gel stained with ethidium bromide.
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Figure 7: Effect of the extension time on DNA amplification from blood (endogenous targets) or crude soil extract. Two targets of the human CCR5 and DNMT genes, 1.1 kb and 0.5 kb, respectively, were amplified in duplex PCR directly from 1.25% to 10% human blood with KlenTaq 10, Taq 22, or w.t. Taq (lanes 1–4, A). Control reactions (lanes C) contained 10 ng DNA and no blood. In (B), the same targets were amplified with the three enzymes from 10 ng human DNA, in the presence of 0.5–20% crude soil extract (lanes 1–5) (Note the shift in the range of the soil extract used with the w.t. enzyme). Control reactions (lanes C) contained no soil extract. Two identical samples of each reaction were amplified with 4 min or 2 min extension time in a no ramp-time PCR cycle. Lanes M, DNA standards ladder. The amplified products were analyzed in a 2% agarose gel stained with ethidium bromide.

Mentions: As some of the known PCR inhibitors found in blood and soil did not show a predominant effect on w.t. Taq activity in the non-PCR assay described above, we looked for another possible way some of these inhibitors may affect DNA amplification. One can assume that in crude, especially blood-containing PCR samples, the DNA template is less available, as it is likely to be bound at least by proteins. This in turn could affect the speed or processivity of the enzyme. Or, alternatively, the enzyme speed could be directly reduced by some inhibitors. Therefore, we examined whether a longer extension time could compensate for lowed speed. DNA amplification of identical samples, containing varying concentrations of a PCR inhibitor, was performed in a no ramp-time PCR cycle, using differing extension times. In the experiment shown in Figure 7, two human targets were amplified in duplex PCR directly from blood (Figure 7A), and purified human DNA was used to amplify the same targets in the presence of a crude soil extract (Figure 7B). We found that doubling the extension time from 2 min to 4 min, facilitated the performance of w.t. Taq, which was able to amplify to some extent the shorter target in 5% blood (the enzyme was not functional with the longer target). In the soil samples series, the w.t. enzyme was only functional in the control reactions containing no soil and a longer extension time did not help, while the mutant enzymes shifted their performance to a higher soil concentration when the extension time was increased. We also observed some reverse correlation between the target size and enzyme performance at a given extension time and concentration of soil inhibitors. In control reactions lacking inhibitors both targets were efficiently amplified regardless of the extension time, which shows that the target ‘difficulty’ was not an issue.

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

Kermekchiev MB, Kirilova LI, Vail EE, Barnes WM - Nucleic Acids Res. (2009)

Effect of the extension time on DNA amplification from blood (endogenous targets) or crude soil extract. Two targets of the human CCR5 and DNMT genes, 1.1 kb and 0.5 kb, respectively, were amplified in duplex PCR directly from 1.25% to 10% human blood with KlenTaq 10, Taq 22, or w.t. Taq (lanes 1–4, A). Control reactions (lanes C) contained 10 ng DNA and no blood. In (B), the same targets were amplified with the three enzymes from 10 ng human DNA, in the presence of 0.5–20% crude soil extract (lanes 1–5) (Note the shift in the range of the soil extract used with the w.t. enzyme). Control reactions (lanes C) contained no soil extract. Two identical samples of each reaction were amplified with 4 min or 2 min extension time in a no ramp-time PCR cycle. Lanes M, DNA standards ladder. The amplified products were analyzed in a 2% agarose gel stained with ethidium bromide.
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Figure 7: Effect of the extension time on DNA amplification from blood (endogenous targets) or crude soil extract. Two targets of the human CCR5 and DNMT genes, 1.1 kb and 0.5 kb, respectively, were amplified in duplex PCR directly from 1.25% to 10% human blood with KlenTaq 10, Taq 22, or w.t. Taq (lanes 1–4, A). Control reactions (lanes C) contained 10 ng DNA and no blood. In (B), the same targets were amplified with the three enzymes from 10 ng human DNA, in the presence of 0.5–20% crude soil extract (lanes 1–5) (Note the shift in the range of the soil extract used with the w.t. enzyme). Control reactions (lanes C) contained no soil extract. Two identical samples of each reaction were amplified with 4 min or 2 min extension time in a no ramp-time PCR cycle. Lanes M, DNA standards ladder. The amplified products were analyzed in a 2% agarose gel stained with ethidium bromide.
Mentions: As some of the known PCR inhibitors found in blood and soil did not show a predominant effect on w.t. Taq activity in the non-PCR assay described above, we looked for another possible way some of these inhibitors may affect DNA amplification. One can assume that in crude, especially blood-containing PCR samples, the DNA template is less available, as it is likely to be bound at least by proteins. This in turn could affect the speed or processivity of the enzyme. Or, alternatively, the enzyme speed could be directly reduced by some inhibitors. Therefore, we examined whether a longer extension time could compensate for lowed speed. DNA amplification of identical samples, containing varying concentrations of a PCR inhibitor, was performed in a no ramp-time PCR cycle, using differing extension times. In the experiment shown in Figure 7, two human targets were amplified in duplex PCR directly from blood (Figure 7A), and purified human DNA was used to amplify the same targets in the presence of a crude soil extract (Figure 7B). We found that doubling the extension time from 2 min to 4 min, facilitated the performance of w.t. Taq, which was able to amplify to some extent the shorter target in 5% blood (the enzyme was not functional with the longer target). In the soil samples series, the w.t. enzyme was only functional in the control reactions containing no soil and a longer extension time did not help, while the mutant enzymes shifted their performance to a higher soil concentration when the extension time was increased. We also observed some reverse correlation between the target size and enzyme performance at a given extension time and concentration of soil inhibitors. In control reactions lacking inhibitors both targets were efficiently amplified regardless of the extension time, which shows that the target ‘difficulty’ was not an issue.

Bottom Line: 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.Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t.Taq polymerase as compared to the mutant enzymes.

Affiliation: DNA Polymerase Technology Inc., Saint Louis, MO 63104, USA. milko@klentaq.com

ABSTRACT

Background: 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.

View Similar Images In: Results  - Collection
View Article: Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2655666&rFormat=json&query=null&req=5