Insertion sequence-based cassette PCR:: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA
Insertion sequence-based cassette PCR:: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA
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DOI：
10.1007/s00253-008-1463-9
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发表时间：
2008-06-01
影响因子：
5
通讯作者：
Tsuda, Masataka
中科院分区：
文献类型：
--
作者：
Fuchu, Genki;Ohtsubo, Yoshiyuki;Tsuda, Masataka
γ - 六氯环己烷（γ - HCH）是一种高度氯化的农药，已造成严重的环境问题。基于目前分离出的几种γ - HCH降解细菌菌株中，插入序列IS6100与γ - HCH降解的lin基因频繁出现的关联性，利用从受六氯环己烷污染的土壤中提取的DNA样本，通过巢式聚合酶链反应（PCR）技术扩增了两侧为两个IS6100拷贝的DNA片段。获得了4个不同的DNA片段，大小分别为6.6、2.6、1.6和1.3 kb，其中3个携带lin基因：6.6 kb的片段携带linD、linE以及linR；2.6 kb的片段呈现linF的截短形式；1.6 kb的片段携带linB。我们这种被称为基于插入序列（IS）的盒式PCR的方法，成功地从受六氯环己烷污染的土壤中分离出lin基因，无需培养宿主细胞，并且可用于不依赖培养分离其他由其他IS元件界定的功能基因。
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.