DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions.

ABSTRACT

Pyrophosphorolysis by bacteriophage T7 DNA polymerase leads to the degradation of specific dideoxynucleotide-terminated fragments on DNA sequencing gels. This reaction can be prevented by pyrophosphatase. It is also inhibited by a high concentration of dNTPs; only the dNTP complementary to the next base in the template is an effective inhibitor, suggesting the formation of a stable polymerase-primer-template-nucleotide complex despite the absence of a 3' hydroxyl group on the primer. The use of pyrophosphatase, a genetically modified T7 DNA polymerase that lacks exonuclease activity, and Mn2+ rather than Mg2+ to eliminate discrimination between dideoxynucleotides and deoxynucleotides (Tabor, S., and Richardson, C. C. (1989) Proc. Nat. Acad. Sci. U. S. A. 86, 4076-4080) generates bands of uniform intensity on a DNA sequencing gel. Uniform band intensities simplify the analysis of a DNA sequence, particularly with automated procedures. For example, when genomic DNA is sequenced directly, heterozygotic sequences are readily detected because their bands have half the intensity of homozygotic sequences. A procedure for automated DNA sequencing is described that exploits the uniformity. A single reaction with a single labeled primer is carried out using four different ratios of dideoxynucleotides to deoxynucleotides; after gel electrophoresis in a single lane, the sequence is determined by the relative intensity of each band.