Format

Send to

Choose Destination
See comment in PubMed Commons below
Electrophoresis. 2008 Apr;29(7):1467-72. doi: 10.1002/elps.200700699.

Interaction of humic acids with human DNA: proposed mechanisms and kinetics.

Author information

1
Department of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Split, Croatia. dsutlov@kbsplit.hr

Abstract

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.

PMID:
18318449
DOI:
10.1002/elps.200700699
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Loading ...
    Support Center