title: DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.
abstract: The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.
authors.affiliation: Department of Biology, University of Louisville, Kentucky 40292.
authors.forename: R J
authors.initials: RJ
authors.lastname: Steffan
authors.forename: R M
authors.initials: RM
authors.lastname: Atlas
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D005818:Genetic Engineering; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011237:Predictive Value of Tests; D011549:Pseudomonas / Q000235:genetics / Q000302:isolation & purification; D012988:Soil Microbiology
chemical_list: D015342:DNA Probes; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase