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

Journal Article · Wed Dec 01 00:00:00 EST 1993 · Applied and Environmental Microbiology; (United States)

OSTI ID:6707211

Erb, R W; Wagner-Doebler, I ^[1]

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1. National Research Center for Biotechnology, Braunschweig (Germany)

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.

• OSTI ID: 6707211
• Journal Information: Applied and Environmental Microbiology; (United States), Vol. 59:12; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

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