Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction - PubMed (original) (raw)

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction

Y L Tsai et al. Appl Environ Microbiol. 1992 Jul.

Abstract

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.

PubMed Disclaimer

Similar articles

Cited by

References

    1. J Bacteriol. 1991 Sep;173(17):5253-5 - PubMed
    1. Appl Environ Microbiol. 1991 Dec;57(12):3456-61 - PubMed
    1. Appl Environ Microbiol. 1990 Feb;56(2):307-14 - PubMed
    1. Nature. 1989 Nov 30;342(6249):485 - PubMed
    1. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436-40 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources