Nucleotide Sequence Analysis of DNA

There are three major obstacles to the analysis of the nucleotide sequence in a DNA molecule starting from a known location in the DNA molecule. First, it is difficult to obtain large quantities of homogeneous DNA. Second, even the smallest DNA molecules contain several thousand nucleotides which make sequence analysis prohibitive. Third, there are no highly base-specific DNAases available for degrading DNA for sequence analysis. We have overcome some of these obstacles; first, by incorporating highly labelled deoxynucleotides into DNA in vitro, small amounts of material can be used for sequence analysis. Second, the nucleotide sequence of DNA molecules can now be determined from the 5′-terminal. Thus, two dodecanucleotide sequences corresponding to the two cohesive ends of λ DNA have been determined¹ and a nona-decanucleotide sequence corresponding to one cohesive end of phage 186 DNA has been completed². So far, our approach is limited to starting the analysis from the 5′-ends of a DNA molecule. A more general approach is being developed for starting the analysis from other selected parts of a DNA molecule with the use of specifically designed primers.