[23] The Use of the T4 DNA Polymerase in Identification of 3′ Terminal Nucleotide Sequences of Duplex DNA

The T4 DNA polymerase can be used to determine the sequence of several nucleotides at the 3' termini of duplex DNA molecules. The DNA is nonradioactive and the single deoxynucleoside triphosphate is labeled in the α-position with ³²P. The T4 DNA polymerase attacks the 3' terminus of the DNA and sequentially releases mononucleotides. Aside from its utility in sequencing, the specific labeling of 3' termini of DNA by means of the T4 polymerase may prove to be as useful as has been the labeling of 5' termini by means of polynucleotide kinase. For example, it would be possible to exchange the two 3'-terminal nucleotides of a DNA molecule with labeled nucleotides. This reaction would introduce no net covalent changes into the molecule, yet each strand would contain a 3' terminal label. It is also possible, by using one triphosphate that is radioactive and one that is nonradioactive, to incorporate a label into one strand, but not into the other.