Strategies for sequencing double-stranded DNA

For sequencing an entire plasmid or virus DNA one can simply begin by cleaving a sample of the DNA into fragments, preferably using a restriction enzyme which yields protruding 5 -ends for ease of labelling. Table 5.II. lists fifteen restriction enzymes which make 

Restriction endonucleases which cleave DNA into fragments readily end-labelled with Trpolynucleotide kinase and y[HP]-ATP or with DNA polymerase and 

Restriction enzyme Sequence recognized Fragment end-structure after cleavage 

Restriction enzymes which cleave double-stranded DNA, making staggered cuts which leave the 5 -end of each strand extended. Double-stranded DNA with protruding 5 -ends is more efficiently phosphorylated by polynucleotide kinase than DNA with flush or recessed 5 -ends. The fore-shortened 3 -ends can also be labelled by extending them with 32P-labelled nucleoside triphosphates complementary to bases in the extended template strand using DNA polymerase. Y—pyrimidine nucleotide, R—purine nucleotide, N—any nucleotide. A complete list of commercially obtainable restriction endonucleases is given in Appendix III. 

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