Insertion sequence-based cassette PCR: Cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA

Genki Fuchu, Yoshiyuki Ohtsubo, Michihiro Ito, Ryo Miyazaki, Akira Ono, Yuji Nagata, Masataka Tsuda

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

γ-Hexachlorocyclohexane (γ-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for γ-HCH degradation in several γ-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.

Original languageEnglish
Pages (from-to)627-632
Number of pages6
JournalApplied Microbiology and Biotechnology
Volume79
Issue number4
DOIs
Publication statusPublished - 2008 Jun 1

Keywords

  • Insertion sequence
  • Soil DNA
  • γ-Hexachlorocyclohexane

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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