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Titolo:
Purification and characterization of a common soil component which inhibits the polymerase chain reaction
Autore:
Watson, RJ; Blackwell, B;
Indirizzi:
Agr & Agri Food Canada, Res Branch, Ottawa, ON K1A 0C6, Canada Agr & Agri Food Canada Ottawa ON Canada K1A 0C6 ttawa, ON K1A 0C6, Canada
Titolo Testata:
CANADIAN JOURNAL OF MICROBIOLOGY
fascicolo: 7, volume: 46, anno: 2000,
pagine: 633 - 642
SICI:
0008-4166(200007)46:7<633:PACOAC>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
ORGANIC-MATTER; BACTERIAL-DNA; REACTION AMPLIFICATION; ENVIRONMENTAL-SAMPLES; DIRECT EXTRACTION; MICROBIAL DNA; RAPID METHOD; PLASMID DNA; SEDIMENTS; SEPARATION;
Keywords:
fulvic acid; humic acid; PCR inhibitor; soil DNA; soil microorganisms;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
41
Recensione:
Indirizzi per estratti:
Indirizzo: Watson, RJ Agr & Agri Food Canada, Res Branch, Ottawa, ON K1A 0C6, Canada Agr & Agri Food Canada Ottawa ON Canada K1A 0C6 1A 0C6, Canada
Citazione:
R.J. Watson e B. Blackwell, "Purification and characterization of a common soil component which inhibits the polymerase chain reaction", CAN J MICRO, 46(7), 2000, pp. 633-642

Abstract

DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soilfractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material becamesoluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 20/09/20 alle ore 07:55:42