Ks. Mohan et Sk. Walia, DETECTION OF SOLUBLE METHANE MONOOXYGENASE PRODUCING METHYLOSINUS-TRICHOSPORIUM OB3B BY POLYMERASE CHAIN-REACTION, Canadian journal of microbiology, 40(11), 1994, pp. 969-973
The soluble methane monooxygenase (sMMO) enzyme complex of methanotrop
hs cometabolizes haloaliphatic compounds such as trichloroethylene. Tw
o 18-mer oligonucleotides as primary primers and a nested primer of th
e same length were selected to amplify specific DNA sequences of the s
MMO gene cluster using polymerase chain reaction (PCR). Two DNA fragme
nts of sizes 270 and 400 base pairs were obtained when purified DNA fr
om the methanotroph Methylosinus trichosporium OB3b was used as templa
te. The primers were specific for sMMO sequences of M. trichosporium,
since none of the 13 bacterial isolates screened yielded the expected
length of PCR-amplified DNA fragments. The detection limit of the PCR
method was 5 x 10(2) cells of M. trichosporium. The sMMO sequences wer
e successfully amplified in groundwater (containing native microbial p
opulation) when seeded with M. trichosporium, FP1 sense (5'-ATGTCCAGCG
CTCATAAC-3'), RP1 antisense (5'-TCAGATCTCGGTCAGGGC-3'), FP2 sense nest
ed (5'GCCATCATCGGTCAGGGC-3'), and FP2 sense nested (5'-GCCATCATCGAGGAC
ATC-3').