The Determination of Nucleotide in DNA and RNA

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 

In methods developed by Sanger s group, DNA of sequence complementary to the template is synthesized by a DNA polymerase-catalysed elongation of a unique primer. The elongation is terminated at a specific type of base (A, G, C, or T) by one of the following methods  

For simplicity, only means of generation of the fragments terminated by A are illustrated. For further details see text. 

By use of these methods, the nucleotide sequence of several hundred fragments of various genomes, including the complete sequence of several DNA viruses, have been determined. Programmes for handling sequence data by computer have been developed.  

A method analogous to that for DNA has been applied to RNA, the 5 -terminally-labelled RNA being partially hydrolysed by alkali or base specific ribonucleases. The scope of this method has been extended by use of the non-specific endo-nuclease Pi and the ribonuclease from Physarium poiy cepharum which cleaves all but CpN linkages, to distinguish between oligonucleotides with Up or Cp 3 termini generated by RNase A from pancreas or RNase from Bacillus cereus.  

---