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


CONCLUSIONS
The modified procedure with increased Taq polymerase concentration should allow more effective QRT-PCR analysis in samples containing HA.
Sutlović
Davorka
D
Department of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Spinciceva 1, 21000 Split, Croatia. dsutlov@kbsplit.hr
Definis Gojanović
Marija
M
Andelinović
Simun
S
Gugić
Dijana
D
Primorac
Dragan
D
D001842:Bone and Bones; D017523:Croatia; D004305:Dose-Response Relationship, Drug; D005826:Genetics, Medical; D006801:Humans; D006812:Humic Substances; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019914:Taq Polymerase / Q000378:metabolism*
D006812:Humic Substances; D019914:Taq Polymerase