Sutlović Davorka, Definis Gojanović Marija, Andelinović Simun, Gugić Dijana, Primorac Dragan
Department of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Spinciceva 1, 21000 Split, Croatia.
Croat Med J. 2005 Aug;46(4):556-62.
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.
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.
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.
The modified procedure with increased Taq polymerase concentration should allow more effective QRT-PCR analysis in samples containing HA.
研究腐殖酸（HA）对定量实时聚合酶链反应（QRT-PCR）抑制的剂量反应效应，以及Taq聚合酶增量在防止古代骨骼提取的DNA中HA抑制方面的效率。
从骨样本中分离DNA，并使用ABI PRISM 7000仪器通过实时5'核酸外切酶检测法（TaqMan）进行DNA定量。
添加10 - 75 ng合成HA可抑制QRT-PCR，而添加100 ng合成HA则完全抑制QRT-PCR。每次测定添加1.25单位（U）的Taq聚合酶似乎是克服HA抑制的最佳量。当含有腐殖物质的粗DNA提取物通过QRT-PCR进行定量，每次测定添加1.25单位（U）额外的Taq聚合酶时，可获得最佳结果。
增加Taq聚合酶浓度的改良程序应能使含有HA的样本进行更有效的QRT-PCR分析。
