Primers That Target Functional Genes of Organohalide-Respiring Bacteria

Halogenated organic hydrocarbons are problematic environmental pollutants that can be reductively dehalogenated by organohalide-respiring bacteria (OHRB) in anoxic environments. This energy-conserving process is mediated by reductive dehalogenases (RDases). To amplify the diversity of reductive dehalogenase-encoding genes, degenerate primers have been designed, most of which target the conserved regions of the encoded protein sequences of the catalytic subunit, RdhA. In addition, specific primer sets have been developed and widely used to quantify and characterise OHRB and the reductive dehalogenase homologous (rdh) genes in the environment. The specific primers have been applied to multiple molecular techniques including regular and quantitative PCR (qPCR), Southern blot hybridisation, terminal restriction fragment length polymorphism (T-RFLP) and reverse transcriptase PCR (RT-PCR). The hunt for novel rdhA genes has benefited greatly from next-generation sequencing techniques, including primer-dependent amplicon sequencing and primer-independent metagenomic analyses. This chapter provides an overview of most primers targeting RDase-encoding genes described to date and their applications, and it discusses the developing trend of leveraging primer-(in)dependent techniques for better understanding of OHRB and their RDase gene pools