An Improved DNA Isolation Method for Metagenomic Analysis of the Microbial Flora of the Human Intestine

Hidetoshi Morita, Tomomi Kuwahara, Kenshiro Ohshima, Hiroyuki Sasamoto, Kikuji Itoh, Masahira Hattori, Tetsuya Hayashi, Hideto Takami

Research output: Contribution to journalArticlepeer-review

66 Citations (Scopus)

Abstract

The efficiency with which lysis of five strictly anaerobic and six facultatively anaerobic bacterial species, all well-known human colonic commensals, were lysed was tested using a reference method for general metagenomic analysis and an improved method that involves higher levels of lysozyme and proteinase K, as well as the addition of achromopeptidase. Ten species were lysed with an efficiency of >80% by the reference method, while the lytic efficiency for Clostridium ramosum JCM 1298T was <50%. The lytic efficiency of the improved method for C. ramosum JCM 1298T was 82.5%. Similarly, five samples of human feces were tested with these methods, as well as with the QIAamp DNA stool mini kit. Although the efficiency of lysis of the microbes recovered from the fecal samples fluctuated depending on the sample in the cases of the reference method (13.3-84.6%) and QIAamp DNA stool mini kit (38.8-69.2%), the improved method gave stable and high-level lysis (>90%) for all the fecal samples. Accordingly, since the DNA samples isolated by the improved method can reflect nearly true genomic information in the microbial flora, our improved method should be applicable to metagenomic analyses, not only for bacteria in the human intestine but also for bacteria in other environments.

Original languageEnglish
Pages (from-to)214-222
Number of pages9
JournalMicrobes and Environments
Volume22
Issue number3
DOIs
Publication statusPublished - 2007
Externally publishedYes

Keywords

  • DNA isolation
  • human feces
  • metagenomics
  • microbial flora

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Soil Science
  • Plant Science

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