53] Determination of fragment order through partial digests and multiple enzyme digests

Abstract

Publisher Summary This chapter focuses on determination of fragment order through partial digests and multiple enzyme digests. The chapter describes the basic principles of two techniques for fragment ordering: analysis of partial digestion products and multiple enzyme digestion. Ordering DNA fragments by partial endonuclease digestion is analogous to a sequencing technique for RNA. Multiple enzyme digestion for ordering DNA fragments employs an approach used for sequencing proteins and RNA. For DNA, the cleavage products of one endonuclease are characterized with respect to size and are then digested with a second endonuclease. Analysis of the resultant double-digestion products establishes the relationship between the cleavage sites of the two enzymes. The chapter presents a model study that develops a physical map of the SV40 genome. The chapter describes the procedures for digestion of DNA with endonuclease and for analysis of cleavage products. Ordering fragments through analysis of partial digestion products is illustrated for the two sets of SV40 DNA fragments produced by cleavage with HincII and Hind lII. Taq I and Barn H1 are used in multiple enzyme digestions with HincII and Hind lII to generate a complex physical map that includes the cleavage sites for all four enzymes.