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Nucleic Acids Research, 1994, Vol. 22, No. 12 2399-2403
© 1994

METHODS

The ‘endo-blue method’ for direct cloning of restriction endonuclease genes in E.coli

Alexey Fomenkov, Jian-ping Xiao, Deborah Dila, Elisabeth Raleigh and Shuang-yong Xu*

New England Biolabs Inc. 32 Tozer Road, Beverly, MA 01915, USA

*To whom correspondence should be addressed

Received January 5, 1994. Revised May 8, 1994. Accepted May 8, 1994.

A new E.coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methyla-tion-dependent restriction systems (McrA–;, McrBC–, Mrr–;). This strain has been used to clone restriction endonuclease genes directly into E.coli. When E.coli cells are not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response. The SOS-induced cells form blue colonies on indicator plates containing X-gal. Using this method the genes coding for the thermostable restriction enzymes TaqI (5'TCGA3') and Tth111I (5'GACNNNGTC3') have been successfully cloned in E.coli. The new strain will be useful to clone other genes involved in DNA metabolism.


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