An improved protocol with a highly degenerate primer targeting copper-containing membrane-bound monooxygenase genes for community analysis of methane- and ammonia-oxidizing bacteria

FEMS Microbiol Ecol. 2017 Mar;93(3):fiw244. doi: 10.1093/femsec/fiw244. Epub 2016 Dec 8.


The copper-containing membrane-bound monooxygenase (CuMMO) family comprises key enzymes for methane or ammonia oxidation: particulate methane monooxygenase (PMMO) and ammonia monooxygenase (AMO). To comprehensively amplify CuMMO genes, a two-step PCR strategy was developed using a newly designed tagged highly degenerate primer (THDP; degeneracy = 4608). Designated THDP-PCR, the technique consists of primary CuMMO gene-specific PCR followed by secondary PCR with a tag as a single primer. No significant bias in THDP-PCR amplification was found using various combinations of template mixtures of pmoA and amoA genes, which encode key subunits of the pMMO and AMO enzymes, respectively, from different microbes. THDP-PCR was successfully applied to nine different environmental samples and revealed relatively high contents of complete ammonia oxidation (Comammox)-related bacteria and a novel group of the CuMMO family. The levels of freshwater cluster methanotrophs obtained by THDP-PCR were much higher than those obtained by conventional methanotroph-specific PCR. The THDP-PCR strategy developed in this study can be extended to other functional gene-based community analyses, particularly when the target gene sequences lack regions of high consensus for primer design.

Keywords: Comammox bacteria; CuMMO; ammonia-oxidizing bacteria; methane-oxidizing bacteria; tagged highly degenerate primer.

MeSH terms

  • Ammonia / metabolism*
  • Bacteria / genetics*
  • Bacteria / metabolism
  • Betaproteobacteria / genetics
  • Copper
  • DNA Primers / genetics
  • Methane / metabolism*
  • Mixed Function Oxygenases / genetics*
  • Oxidation-Reduction
  • Oxidoreductases
  • Oxygenases
  • Polymerase Chain Reaction / methods


  • DNA Primers
  • Ammonia
  • Copper
  • Mixed Function Oxygenases
  • Oxidoreductases
  • Oxygenases
  • methane monooxygenase
  • ammonia monooxygenase
  • Methane