The Determination of Nucleotide Sequences in DNA and RNA

The Determination of Nucleotide Sequences In DNA and RNA.—DNA. The methods developed by Sanger and co-workers, reported in the last volume, which use specific chain-terminators (dideoxyribonucleoside triphosphates) have proved particularly valuable when applied to single-stranded DNA. However, the scope of the method was limited to naturally occurring single-stranded DNA or to duplex DNA in which strands could be readily separated. 

Suitable vectors have been derived from the bacteriophage M13 which, except in its replicating form (RF), contains single-stranded circular DNA. The derivative M13 mpl, produced by insertion of part of the Escherichia coli lac operon, was mutated to give a single site for the restriction endo nuclease EcoRl within the gene for /8-D-galactosidase. This enzyme produces a blue colour when the hosts of this 

Digestion vith restriction enzyme. Addition of EcoRl linker and digestion vith EcoRl. (see Fig. 2) 

Extensitm of the priaier with random terminaticm by the four ddNTPs to generate fragments of random size 300 nucleotides) for separation on gels. 

In order to sequence the inserted DNA by the dideoxy method , a suitable primer adjacent to the inserted DNA is required. One such primer, 96 nucleotides long, was obtained by introduction of a second EcoRl site in Ml3 mp2 to generate M13 mp2962. Annealing of this primer to DNA from a randomly selected M13 mp2 recombinant permits sequencing of approximately 200 bases to the 3 side of the 96-nucleotide primer. The method has been further refined by synthesis of a smaller (30 base pair) primer and its cloning into the plasmid vector pBR322 for amplification as required. With this primer, or the commercially available 12 base 

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The Determination of Nucleotide Sequences in DNA and RNA.—DNA. By electro phoresis on polyacrylamide gels it is possible to resolve DNA strands into bands... 

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