Interaction of humic acids with human DNA: proposed mechanisms and kinetics.
Human DNA quantification by quantitative real-time PCR (QRT-PCR) has gained great importance in forensic DNA and ancient DNA studies. However, in such samples, DNA quantification is impaired by the frequently present humic acid (HA). We have previously shown that the addition of synthetic HA inhibits QRT-PCR. In this study we investigated the possible mechanisms of HA interaction with human DNA, and kinetics of QRT-PCR inhibition. In QRT-PCR with pure human DNA and no HA added, VMAX was 40. With DNA sample containing 4 microg/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 75 to 87 degrees C and inhibits DNase I-mediated DNA cleavage, most probably affecting the enzyme's activity.
Sutlovic
Davorka
D
Department of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Split, Croatia. dsutlov@kbsplit.hr
Gamulin
Stjepan
S
Definis-Gojanovic
Marija
M
Gugic
Dijana
D
Andjelinovic
Simun
S
D004247:DNA / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D006812:Humic Substances; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet
D006812:Humic Substances; D004247:DNA