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Croat Med J. 2005 Aug;46(4):556-62.

Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid.

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  • 1Department of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Spinciceva 1, 21000 Split, Croatia. dsutlov@kbsplit.hr

Abstract

AIM:

To investigate the dose-response effect of humic acid (HA) on the quantitative real time polymerase chain reaction (QRT-PCR) inhibition and the efficiency of Taq polymerase increment in preventing inhibition by HA in DNA extracted from ancient bones.

METHODS:

DNA was isolated from bone samples and DNA quantification was conducted with the real-time 5' exonuclease detection assay (TaqMan), using the ABI PRISM 7000 instrument.

RESULTS:

The addition 10-75 ng of synthetic HA inhibited QRT-PCR, whereas the addition of 100 ng of synthetic HA completely inhibits QRT-PCR. The addition of 1.25 Unit (U) of Taq polymerase per assay appeared to be the optimum amount in overcoming the HA inhibition. The best results were obtained when crude DNA extracts containing humic substances were quantified by QRT-PCR with the addition of 1.25 Unit (U) of extra Taq polymerase per assay.

CONCLUSION:

The modified procedure with increased Taq polymerase concentration should allow more effective QRT-PCR analysis in samples containing HA.

PMID:
16100758
[PubMed - indexed for MEDLINE]
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