Analysis of Nucleotide Sequences at 3' Termini of Duplex Deoxyribonucleic Acid with the Use of the T4 Deoxyribonucleic Acid Polymerase

The T4 DNA polymerase catalyzes extensive exonucleolytic degradation of linear duplex DNA starting at the 3' ends. However, the enzyme will release only a few nucleotides from each DNA molecule if the degradation is run in the presence of a single deoxynucleoside triphosphate. Several experiments suggested that the polymerase degrades a chain only until a nucleotide is released which can be replaced by transfer from the triphosphate. Subsequent reaction consists only of alternating removal and replacement of that nucleotide, without net degradation of the DNA. Because only a few nucleotides are released in the presence of a single triphosphate, it seemed possible that the polymerase could be used for analysis of nucleotide sequences at 3' termini of duplex DNA. This possibility was verified by the analysis of the 3'-terminal sequence of fragments of bacteriophage T7 DNA produced by endonuclease R, the Hemophilus influenzae restriction endonuclease. This terminal sequence had been previously identified by Kelly and Smith (J. Mol. Biol., 51, 393 (1970)). The method used in this analysis, which should also be applicable to other DNAs, was to degrade 32P-labeled DNA by the T4 polymerase in the presence of each of the four triphosphates. The nucleotides released in each case were identified and quantitatively determined by thin layer chromatography, and were consistent with the previously determined terminal sequence.