Development of degenerate and specific PCR primers for the detection and isolation of known and putative chloroethene reductive dehalogenase genes

J Microbiol Methods. 2004 Jan;56(1):107-18. doi: 10.1016/j.mimet.2003.09.019.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacteria, Anaerobic / enzymology*
  • Bacteria, Anaerobic / genetics
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers / chemistry*
  • DNA Primers / genetics*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • DNA, Bacterial / isolation & purification*
  • Genes, Bacterial
  • Molecular Sequence Data
  • Oxidoreductases / genetics*
  • Polymerase Chain Reaction / methods
  • Sequence Alignment
  • Sequence Analysis, DNA


  • DNA Primers
  • DNA, Bacterial
  • Oxidoreductases
  • tetrachloroethene dehalogenase