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

  • Applied Genetics and Molecular Biotechnology
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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.

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Acknowledgements

This work was supported by Grant-in-Aids from Ministry of Education, Culture, Sports, Science, and Technology and The Ministry of Agriculture, Forestry, and Fisheries (HC-07-2323), Japan.

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Authors and Affiliations

  1. Department of Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan

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

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  1. Genki Fuchu
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  2. Yoshiyuki Ohtsubo
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  3. Michihiro Ito
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  4. Ryo Miyazaki
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  5. Akira Ono
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  6. Yuji Nagata
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  7. Masataka Tsuda
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Correspondence to Yuji Nagata.

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Fuchu, G., Ohtsubo, Y., Ito, M. et al. Insertion sequence-based cassette PCR: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA. Appl Microbiol Biotechnol 79, 627–632 (2008). https://doi.org/10.1007/s00253-008-1463-9

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  • DOI: https://doi.org/10.1007/s00253-008-1463-9

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