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Influence of humic acid on the success of DNA quantification and typing by Real time, PCR and PCR methods

The identification process of dead bodies or human remains is now days conducted in numerous fields of forensic science, archeology and other judicial cases. A particular problem is the isolation and DNA typing of human remains found in mass graves, due to the degradation process, as well as post mortal DNA contamination with bacteria, fungi, humic acids (HA), metals etc. In this study we investigated the influence of humic acid on the success of DNA extraction, quantification and typing by Real time - PCR and PCR methods. It was reported that the humic acid if present in the amplification reaction mix inhibited the DNA amplification, but the addition of 50 mg PVPP to the reaction mixture before extraction, appeared to be optimum in overcoming that inhibition. It was investigated the dose-response effect of humic acid 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. 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 quantified by QRT-PCR, with adding of 1.25 Unit (U) of extra Taq polymerase per assay. It was investigated the possible mechanisms of HA interaction with human DNA, and kinetics of QRT-PCR inhibition were investigated. In QRT-PCR with pure human DNA and no HA added, VMAX was 40. With DNA sample containing 4 g/ml of HA, VMAX was 30.30 while the addition of extra Taq polymerase to the same sample changed VMAX into 38.91, amplifying between 80 and 90% of input DNA. The KM/VMAX ratio in all the samples remained constant, indicating that the mechanism of HA inhibition of QRT-PCR is uncompetitive by nature. Moreover HA shifts the human DNA melting temperature point (Tm) from 75oC to 87oC and inhibits DNase I mediated DNA cleavage, most probably affecting the enzyme’s activity.

Forensic genetics, humic acid, PCR methods, PCR inhibition

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