DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions.
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.
Tabor
S
S
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Richardson
C C
CC
D000818:Animals; D001322:Autoanalysis; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D004330:Drosophila / Q000235:genetics; D006580:Genetic Carrier Screening; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000494:pharmacology; D013604:T-Phages / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D010710:Phosphates; D013942:Thymine Nucleotides; C037773:2',3'-dideoxyadenosine triphosphate; D008345:Manganese; C037406:2',3'-dideoxycytidine 5'-triphosphate; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D011755:Pyrophosphatases; D008274:Magnesium; C017216:2',3'-dideoxythymidine triphosphate