[24] Enzymatic Methods for Sequence Analysis Applied to DNA Restriction and Methylation Sites

Restriction enzymes are site-specific endonucleases produced by various bacteria, bacterial plasmids, and viruses. In those cases where they produce double-stranded cleavage of DNA within their recognition sites, sequence analysis of the site is equivalent to determination of the 5'- and 3'-terminal bases of the restricted DNA. This chapter describes a method for limited sequencing of the 5' terminus of DNA molecules based on enzymatic cleavage of successively larger oligonucleotides. This procedure was developed especially for analysis of the end sequence of small restriction endonuclease fragments but should be more generally applicable to any small DNA molecule. Every host that produces a restriction endonuclease also produces a companion DNA modification enzyme, usually a methylase, with identical or similar site recognition. This enzyme serves to modify and protect the restriction sites of the host chromosome. The chapter concludes by describing a procedure for partial analysis of methylation sites.