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Tags : Real time PCR   PCR inhibition   humic acid  more

Tags : Real time PCR   PCR inhibition   humic acid   quantification   Taq polymerase  less

Reference : 1. Sutlovic D, Definis Gojanovic M, Andjelinovic S, Gugic D, Primorac D. Taq Polymerase Reverses In    more

Reference : 1. Sutlovic D, Definis Gojanovic M, Andjelinovic S, Gugic D, Primorac D. Taq Polymerase Reverses Inhibition of Quantitative Real Time Polymerase Chain Reaction by Humic Acid. Croat Med J 2005; 46(4):556-62. 2. Sutlovic D, Definis Gojanovic M, Andjelinovic S. Rapid Extraction of Human DNA containing humic acid. Croat Chem Acta 2007; 80(1) : 117-20. 3. Sutlovic, Davorka; Gamulin, Stjepan; Definis-Gojanovic, Marija; Gugic, Dijana; Andjelinovic, Simun. Interaction of Humic Acids with Human DNA: Proposed Mechanisms and Kinetics. Electrophoresis 2008;29:7:1467-72.    less

Abstract : Aim. To investigate the dose-response effect of humic acid (HA) on the Quantification Real Time PC    more

Abstract : Aim. To investigate the dose-response effect of humic acid (HA) on the Quantification Real Time PCR (QRT-PCR) inhibition and the efficiency of Taq polymerase increment in preventing inhibition by HA in DNA extracted from ancient bones. Methods. DNA had been isolated from bone samples and DNA quantification was conducted with the real-time 5’ exonuclease detection assay (TaqMan?), using the ABI PRISM? 7000 instrument from Applied Biosystems, Foster City, CA USA. Results. The addition 10 - 75 ng of synthetic HA (Fluka) can inhibit QRT- PCR while 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 quantificated by QRT-PCR, with adding of 1.25 Unit (U) of extra Taq polymerase per assay. Conclusion. This modified procedure (increment of Taq polymerase) should allow more effective QRT-PCR analysis in samples containing HA. - Slides    less