Conferring new cleavage specificities of restriction endonucleases

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Abstract

This chapter discusses the conferring new cleavage specificities of restriction endonucleases. It describes a set of protocols for completely methylating both plasmid and intact chromosomal DNA. Achilles' cleavage (AC) is a simple, general method for combining the cleavage specificities of restriction endonucleases with the binding specificities of other DNA-binding molecules. With this technique, any restriction site that can be protected from methylation by its cognate methyltransferase (MTase) can be made unique. This chapter also discusses general aspects of the AC process and also contains a protocol for RecA-AC, which is at this time the most general and therefore the most useful of the AC reactions. Restriction enzymes that efficiently cleave DNA at short but specific sequences have played a central role in the development of modern molecular genetics. By dramatically increasing the specificity of these enzymes, AC amplifies the power of conventional recombinant DNA technology, particularly, when it is applied to the physical mapping and precise molecular dissection of multi megabase genomes.

Original languageEnglish (US)
Pages (from-to)321-329
Number of pages9
JournalMethods in Enzymology
Volume216
Issue numberC
DOIs
StatePublished - Jan 1 1992

Bibliographical note

Funding Information:
We are grateful to Dr. John Hanish for helpful discussions, the National Institutes of Health for grant support, and the Howard Hughes Medical Institute for a predoctoral fellowship to S.A.S.

Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.

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