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

Autori
Sutlović, Davorka ; Definis-Gojanović, Marija ; Anđelinović, Šimun ; Gugić, Dijana ; Primorac, Dragan

Izvornik
Croatian medical journal (0353-9504) 46 (2005), 4; 556-562

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
bovine serum-albumin; DNA quantification; PCR amplification; mitochondrial-DNA; bacterial-DNA; microbial DNA; rapid method; ancient DNA; copy number; soil

Sažetak
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.

Izvorni jezik
Engleski

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