Comparative in silico analysis of PCR primers suited for diagnostics and cloning of ammonia monooxygenase genes from ammonia-oxidizing bacteria.
Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study.
Junier
Pilar
P
Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland. pilar.junier@epfl.ch
Kim
Ok-Sun
OS
Molina
Verónica
V
Limburg
Petra
P
Junier
Thomas
T
Imhoff
Johannes F
JF
Witzel
Karl-Paul
KP
D000641:Ammonia / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D020562:Betaproteobacteria / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D020563:Gammaproteobacteria / Q000201:enzymology / Q000235:genetics; D009876:Operon; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment
D017931:DNA Primers; D000641:Ammonia; D010088:Oxidoreductases; C045582:ammonia monooxygenase