skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction

Abstract

It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomaonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namel y, nahC and TodE) were presentmore » was excluded. Thus, for the first time it was possible to directly amplify and detect a chromosomally encoded, single-copy gene from a highly specialized subpopulation of the total microbial community in natural sediments.« less

Authors:
;  [1]
  1. National Research Center for Biotechnology, Braunschweig (Germany)
Publication Date:
OSTI Identifier:
6707211
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology; (United States)
Additional Journal Information:
Journal Volume: 59:12; Journal ID: ISSN 0099-2240
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; BIODEGRADATION; GENETIC CONTROL; DNA; ECOLOGICAL CONCENTRATION; POLYCHLORINATED BIPHENYLS; PSEUDOMONAS; GENES; SEDIMENTS; CONTAMINATION; EVALUATION; PROBES; AROMATICS; BACTERIA; CHEMICAL REACTIONS; CHLORINATED AROMATIC HYDROCARBONS; CONTROL; DECOMPOSITION; HALOGENATED AROMATIC HYDROCARBONS; MICROORGANISMS; NUCLEIC ACIDS; ORGANIC CHLORINE COMPOUNDS; ORGANIC COMPOUNDS; ORGANIC HALOGEN COMPOUNDS; PEST CONTROL; 540220* - Environment, Terrestrial- Chemicals Monitoring & Transport- (1990-); 550400 - Genetics

Citation Formats

Erb, R W, and Wagner-Doebler, I. Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction. United States: N. p., 1993. Web.
Erb, R W, & Wagner-Doebler, I. Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction. United States.
Erb, R W, and Wagner-Doebler, I. 1993. "Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction". United States.
@article{osti_6707211,
title = {Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction},
author = {Erb, R W and Wagner-Doebler, I},
abstractNote = {It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomaonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namel y, nahC and TodE) were present was excluded. Thus, for the first time it was possible to directly amplify and detect a chromosomally encoded, single-copy gene from a highly specialized subpopulation of the total microbial community in natural sediments.},
doi = {},
url = {https://www.osti.gov/biblio/6707211}, journal = {Applied and Environmental Microbiology; (United States)},
issn = {0099-2240},
number = ,
volume = 59:12,
place = {United States},
year = {1993},
month = {12}
}