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

Applied Microbiology and Biotechnology
Genki FuchuMasataka Tsuda

Abstract

gamma-Hexachlorocyclohexane (gamma-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 gamma-HCH degradation in several gamma-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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Citations

Jan 16, 2014·Indian Journal of Microbiology·Rong ChenTian Xie
Apr 17, 2009·Applied Microbiology and Biotechnology·Sho Morimoto, Takeshi Fujii
Mar 4, 2010·Microbiology and Molecular Biology Reviews : MMBR·Rup LalJohn G Oakeshott
Sep 2, 2016·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Michiro TabataYuji Nagata
May 8, 2019·Environmental Microbiology Reports·Yuji NagataMasataka Tsuda

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