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Nucleic Acids Research, 1991, Vol. 19, No. 5 1081-1085
© 1991

MOLECULAR BIOLOGY

PCR with 5-methyl-dCTP replacing dCTP

Kwong Kwok Wong and Michael McClelland*

California Institute of Biological Research 11099 North Torrey Pines Road, La Jolla, CA 92037, USA

* To whom correspondence should be addressed

Received November 26, 1990. Revised January 30, 1991. Accepted January 30, 1991.

When dCTP is replaced by methYl5-dCTP in the polymerase chain reaction some templates cannot be efficiently amplified by Taq polymerase or VentTM polymerase using standard cycling parameters. However, this phenomenon can be overcome by increasing the temperature of the denaturation steps to 100°C, or by adding dlTP to destabilize the m5dC:dG base pairs. Once the block to amplification of m5dC-substituted DNA was overcome, methylated DNA from the ‘superpotylinker’ of the plasmid pSL118O was used as a substrate to check the methyl-sensitivity of a variety of restriction endonucleases. The m5dC-substituted DNAs should also be valuable substrates for defining the specificity of methyl-dependent endonucleases.


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