POLYMERASE CHAIN-REACTION AMPLIFICATION AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS OF 16S RIBOSOMAL-RNA GENES FROM METHANOGENS

For restriction fragment length polymorphism (RFLP) analysis of 16S rR NA genes, the rDNA fragments of 1.5 kb were amplified by polymerase ch ain reaction (PCR) from crude cell lysates of various methanogenic spe cies which were prepared by a combined technique of ultrasonic treatme nt and protease digestion. The PCR products were purified by the polye thylene glycol precipitation method and treated with various restricti on enzymes. The 16S rDNA fragments digested with HaeIII or HhaI gave s pecies-specific RFLP profiles on simplified agarose gel electrophoresi s. 16S rDNA fragments of 0.4 kb from the bulk DNA extracted from mixed populations of anaerobic sludge were also amplified by PCR with a pai r of methanogen-specific primers and cloned directly by the T-A clonin g technique. The cloned 16S rDNAs from recombinants were reamplified b y PCR, and RFLP pattern analysis was performed following digestion wit h HhaI. The PCR-RFLP analysis of 16S rDNA with the present protocol ca n be completed within one day, provided that sufficient amounts of tes t cells are available, and has great promise as a simple and rapid tec hnique for identification of methanogens. A combined method consisting of PCR amplification, direct cloning with T vectors, and RFLP analysi s of 16S rDNA is also useful for rapid estimation of the mixed populat ion structure of methanogens without the need for cultivation and isol ation.