Abstract
Publisher Summary This chapter discusses the conferring new specificities on restriction enzymes that is the cleavage at any predetermined site by combining adapter oligodeoxynucleotide and class-IIS enzyme. Class-II restriction endonucleases (ENases), the most widely used tools in genetic engineering at present, cleave double-stranded (ds) DNA at pre-existing recognition sites, which usually are 4–8 bp long. The number of such recognition sites is quite limited; it would be convenient to have a means for cutting DNA at any predetermined site. Such a method, as adopted for single-stranded (ss) DNA, is described and depends on the use of a specially designed oligodeoxyribonucleotide (oligo) adapter carrying the class-IIS ENase recognition site, which directs the cognant class-IIS ENase to a specific predetermined cleavage site. A specially designed adapter-primer oligo permits one to produce DNA fragments with a predetermined end located between any specified two base pairs of the target DNA. This approach is especially useful for DNA fragments cloned in an ssDNA-generating vector, and it could also complement our other preprogrammed DNA-trimming method. The chapter discusses the dissecting mechanisms of enzyme recognition, endonucleolytic cleavage, and methylation.
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