Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis.

Applied and Environmental Microbiology
B A HalesJ R Saunders

Abstract

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed...Continue Reading

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Citations

Nov 15, 2000·FEMS Microbiology Reviews·W LiesackN P Revsbech
Jan 7, 2003·Applied and Environmental Microbiology·Marcus A HornHarold L Drake
Dec 4, 2003·Environmental Microbiology·Pierre E GalandKim Yrjälä
Mar 10, 2004·Applied and Environmental Microbiology·André-Denis G WrightKellie D Smith
Sep 15, 2005·Environmental Microbiology·Heli JuottonenKim Yrjälä
Dec 8, 2005·Applied and Environmental Microbiology·Duane P MoserT C Onstott
Apr 2, 2008·Applied and Environmental Microbiology·Lisa M GiegJoseph M Suflita
Aug 30, 2008·Environmental Microbiology·Katharina F EttwigMarc Strous
Dec 1, 2002·FEMS Microbiology Ecology·Pierre E GalandKim Yrjälä
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Feb 22, 2011·Applied and Environmental Microbiology·Cassandre Sara LazarLaurent Toffin
Oct 5, 2010·Microbial Ecology·Peng WangChuanlun Zhang
Apr 13, 2011·Astrobiology·Dirk Schulze-MakuchSteven J Hallam
Jan 18, 2012·Applied Microbiology and Biotechnology·Serge Maurice MbadingaBo-Zhong Mu
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Jul 2, 2003·The Journal of General and Applied Microbiology·Motoo UtsumiHiroo Uchiyama

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