Heat-stable DNA polymerase I large fragment resolves hairpin structure in DNA sequencing

A heat-stable large fragment was obtained by subtilisin digestion of DNA polymerase, prepared from Bacillus stearothermophilus. The dideoxy sequencing method, combined with the use of M13 vector has proved to be the most powerful one for obtaining the sequences of large genomes. However, the hairpin structure formed along the single-stranded DNA template often prevents the DNA polymerase from moving on, with the result that no sequence information can be obtained. The heat-stable large fragment that we have obtained has proved to be the most active at 65 degrees C. When the sequencing reaction was carried out at this temperature, the hairpin structure was resolved and the sequencing gels obtained were satisfactory.