Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments.
DNA quantification of soils and sediments is useful for the investigation of microbial communities and for the acquisition of their genomes that are exploited for the production of natural products. However, in such samples DNA quantification is impaired by humic acids (HA). Due to its lack of specificity and sensitivity, UV spectrophotometry cannot be applied. Consequently, fluorimetric assays applying Hoechst (H) 33258 or PicoGreen (PG) are used. Here, we investigated the SYBR Green I (SG) assay, which was also affected by HA, but was found to be 25- and 1.7-fold more sensitive compared to the H 33258 and PG assays, respectively. Spectrophotometric, fluorimetric and quenching studies as well as gel mobility shift assays suggested that the effect of HA on the SG assay was based on an inner filter effect, collisional quenching and binding of SG to HA. As to the latter finding, the standard 6250-fold dilution of the SG reagent was optimised to a 2000-fold dilution. Although the sensitivity of the optimised SG assay was reduced by a factor of 1.3, the interfering effect of HA could be reduced up to 22-fold. A significant reduction of HA interferences by lowering the pH of the assay was not observed. Finally, the performance of the modified SG assay and the corresponding evaluation methods were verified by the determination of DNA recoveries and concentrations of standards and environmental samples in comparison to the PG assay.
Zipper
Hubert
H
Laboratory of Biochemistry, Chair for Interfacial Engineering, University of Stuttgart and Fraunhofer IGB, Nobelstrasse 12, D-70569 Stuttgart, Germany.
Buta
Christiane
C
Lämmle
Katrin
K
Brunner
Herwig
H
Bernhagen
Jürgen
J
Vitzthum
Frank
F
D052160:Benzothiazoles; D001667:Binding, Competitive; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D003959:Diamines; D024202:Electrophoretic Mobility Shift Assay; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D019015:Geologic Sediments / Q000032:analysis*; D006812:Humic Substances / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009930:Organic Chemicals; D011804:Quinolines; D012680:Sensitivity and Specificity; D012987:Soil / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014867:Water / Q000032:analysis*
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D006812:Humic Substances; D009930:Organic Chemicals; C099476:PicoGreen; D011804:Quinolines; D012987:Soil; D014867:Water; C098022:SYBR Green I; D004247:DNA