title: Development of degenerate and specific PCR primers for the detection and isolation of known and putative chloroethene reductive dehalogenase genes.
abstract: Degenerate and specific PCR primers were designed for the detection of chloroethene reductive dehalogenases (CE-RDase), the key enzymes of chloroethene dehalorespiration, based on sequence information of three CE-RDases and three chlorophenol (CP) RDases. For the design of the degenerate primers, seven conserved amino-acid blocks identified with different bioinformatic tools were used. For one block degenerate, primers containing a 5'-consensus clamp region specific for CE-RDases and a 3'-end degenerate core region specific for RDases in general were designed using the Consensus-Degenerate Hybrid Oligonucleotide Primer (CDHOP) design method. Applying the degenerate primers to genomic DNA of Sulfurospirillum multivorans strain K, Dehalobacter restrictus strain PER-K23, and Desulfitobacterium sp. strain PCE1 led to the isolation of the known CE-RDase genes and three new genes encoding putative reductive dehalogenases that cluster with CE-RDases and not with CP-RDases. In addition, primers designed to be specific for the three known CE-RDase genes, namely pceA of S. multivorans, pceA of D. restrictus, and tceA of Dehalococcoides ethenogenes were successfully tested on genomic DNA of different chloroethene-dehalorespiring bacteria. Nested PCR using degenerate primers followed by a PCR with specific primers allowed a sensitive detection of only 10(2) copies per reaction.
authors.affiliation: Laboratory for Environmental Biotechnology, Swiss Federal Institute of Technology Lausanne (EPFL), ENAC-ISTE, Bâtiment CH-B Ecublens, 1015 Lausanne, Switzerland.
authors.forename: Christophe
authors.initials: C
authors.lastname: Regeard
authors.forename: Julien
authors.initials: J
authors.lastname: Maillard
authors.forename: Christof
authors.initials: C
authors.lastname: Holliger
mesh_terms: D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D010088:Oxidoreductases; C112990:tetrachloroethene dehalogenase