A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria)

Abstract

High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS. DNA can subsequently be purified by the usual phenol-chloroform extractions. The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation. The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell.

Methermicoccus shengliensis gen. nov., sp. nov., a thermophilic, methylotrophic methanogen isolated from oil-production water, and proposal of Methermicoccaceae fam. nov.

“…Genomic DNA was isolated and purified by using the method of Marmur (1961) as modified by Jarrell et al (1992), and its G+C content was determined by the thermal-denaturation method (Marmur & Doty, 1962), using a Beckman DU 800 spectrophotometer. Escherichia coli K12 DNA was used as a reference.…”

Isolation and characterization of a new Methanosarcina mazei strain GFJ07 from a mountain forest pond

“…DNA of the isolate was extracted by procedure reported previously (Jarrell et al, 1992). The 16S rDNA was amplified by PCR from genomic DNA with primer Met86F (5'-GCTCAGTAACACGTGG-3') and Met1340R (5'-CGGTGTGTGCAAGGAG-3') (Saengkerdsub et al, 2007).…”

Methanofollis formosanus sp. nov., isolated from a fish pond

“…T and strain ML15 T were obtained as described previously (Jan et al, 1999;Lai et al, 2004;Marmur & Doty, 1962) For phylogenetic analysis, DNA was isolated by the general procedure of Jarrell et al (1992). Approximately 30 ng DNA was used as a template for PCR amplification of an approximately 1300 bp portion of the 16S rRNA gene.…”