TY - JOUR
T1 - Insertion sequence-based cassette PCR
T2 - Cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA
AU - Fuchu, Genki
AU - Ohtsubo, Yoshiyuki
AU - Ito, Michihiro
AU - Miyazaki, Ryo
AU - Ono, Akira
AU - Nagata, Yuji
AU - Tsuda, Masataka
N1 - Funding Information:
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.
PY - 2008/6
Y1 - 2008/6
N2 - 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.
AB - 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.
KW - γ-Hexachlorocyclohexane
KW - Insertion sequence
KW - Soil DNA
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U2 - 10.1007/s00253-008-1463-9
DO - 10.1007/s00253-008-1463-9
M3 - Article
C2 - 18425509
AN - SCOPUS:44649177513
SN - 0175-7598
VL - 79
SP - 627
EP - 632
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 4
ER -