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Titolo:
Effects of humic substances on fluorometric DNA quantification and DNA hybridization
Autore:
Bachoon, DS; Otero, E; Hodson, RE;
Indirizzi:
Georgia Coll, Dept Biol & Environm Sci, Milledgeville, GA 31061 USA Georgia Coll Milledgeville GA USA 31061 Sci, Milledgeville, GA 31061 USA State Univ, Milledgeville, GA 31061 USA State Univ Milledgeville GA USA 31061 e Univ, Milledgeville, GA 31061 USA Dept Marine Sci, UPR, Mayaguez, PR 00981 USA Dept Marine Sci Mayaguez PR USA 00981 ne Sci, UPR, Mayaguez, PR 00981 USA Univ Georgia, Sch Marine Sci, Athens, GA 30602 USA Univ Georgia Athens GAUSA 30602 ia, Sch Marine Sci, Athens, GA 30602 USA
Titolo Testata:
JOURNAL OF MICROBIOLOGICAL METHODS
fascicolo: 1, volume: 47, anno: 2001,
pagine: 73 - 82
SICI:
0167-7012(200110)47:1<73:EOHSOF>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
RIBOSOMAL-RNA; RAPID EXTRACTION; MICROBIAL DNA; PURIFICATION; SEDIMENTS; SOIL; MICROORGANISMS; COMMUNITY; RECOVERY; SAMPLES;
Keywords:
DNA hybridization; Hoechst dye; humics; PicoGreen;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
26
Recensione:
Indirizzi per estratti:
Indirizzo: Bachoon, DS Georgia Coll, Dept Biol & Environm Sci, Milledgeville, GA 31061 USA Georgia Coll Milledgeville GA USA 31061 geville, GA 31061 USA
Citazione:
D.S. Bachoon et al., "Effects of humic substances on fluorometric DNA quantification and DNA hybridization", J MICROB M, 47(1), 2001, pp. 73-82

Abstract

DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities, Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/mul) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80ng/mul of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (< 2.0 mug) of humic substances derived from estuarine waters did not affect the binding of 100 ngof target DNA to nylon membranes. DNA samples containing 1.0 mug of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (< 20 ng/mul) could be used inquantitative membrane hybridization without further purification. (C) 2001Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/09/20 alle ore 23:47:32