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A heat-stable large fragment was obtained by subtilisin digestion of DNA polymerase, prepared fromBacillus stearothermophilus. The dideoxy sequencing method~([1]), combined with the use of M13 vector~([2,3])has proved to be the most powerful one for obtaining the sequences of large genomes. However, the hair-pin structure formed along the single-stranded DNA template often prevents the DNA polymerase frommoving on, with the result that no sequence information can be obtained. The heat-stable large fragmentthat we have obtained has proved to be the most active at 65℃. When the sequencing reaction was car-ried out at this temperature, the hairpin structure was resolved and the sequencing gels obtained weresatisfactory.
A heat-stable large fragment was obtained by subtilisin digestion of DNA polymerase, prepared from Bacillus stearothermophilus. The dideoxy sequencing method ~ ([1]), combined with the use of M13 vector ~ ([2,3]) has proved to be the Most powerful one for obtaining the sequences of large genomes. However, the hair-pin 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. The heat-stable large fragmentThat we have obtained proved to be the most active at 65 ℃. When the sequencing reaction was car-ried out at this temperature, the hairpin structure was resolved and the sequencing gels obtained weresatisfactory.