Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.

S Tabor, C C Richardson
PMCID: PMC305186  PMID: 3474623

Abstract

A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3' to 5' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.

Full text

PDF
4768

Images in this article

4769Image
on p.4769
4769Image
on p.4769
4769Image
on p.4769
4770Image
on p.4770
4770Image
on p.4770

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adler S., Modrich P. T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits. J Biol Chem. 1979 Nov 25;254(22):11605–11614. [PubMed] [Google Scholar]
  2. Ansorge W., Labeit S. Field gradients improve resolution on DNA sequencing gels. J Biochem Biophys Methods. 1984 Dec;10(3-4):237–243. doi: 10.1016/0165-022x(84)90043-5. [DOI] [PubMed] [Google Scholar]
  3. Atkinson M. R., Deutscher M. P., Kornberg A., Russell A. F., Moffatt J. G. Enzymatic synthesis of deoxyribonucleic acid. XXXIV. Termination of chain growth by a 2',3'-dideoxyribonucleotide. Biochemistry. 1969 Dec;8(12):4897–4904. doi: 10.1021/bi00840a037. [DOI] [PubMed] [Google Scholar]
  4. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Engler M. J., Lechner R. L., Richardson C. C. Two forms of the DNA polymerase of bacteriophage T7. J Biol Chem. 1983 Sep 25;258(18):11165–11173. [PubMed] [Google Scholar]
  6. Fischer H., Hinkle D. C. Bacteriophage T7 DNA replication in vitro. Stimulation of DNA synthesis by T7 RNA polymerase. J Biol Chem. 1980 Aug 25;255(16):7956–7964. [PubMed] [Google Scholar]
  7. Gough J. A., Murray N. E. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol. 1983 May 5;166(1):1–19. doi: 10.1016/s0022-2836(83)80047-3. [DOI] [PubMed] [Google Scholar]
  8. Hori K., Mark D. F., Richardson C. C. Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase. J Biol Chem. 1979 Nov 25;254(22):11598–11604. [PubMed] [Google Scholar]
  9. Joyce C. M., Grindley N. D. Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1830–1834. doi: 10.1073/pnas.80.7.1830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kunkel T. A., Eckstein F., Mildvan A. S., Koplitz R. M., Loeb L. A. Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6734–6738. doi: 10.1073/pnas.78.11.6734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lechner R. L., Engler M. J., Richardson C. C. Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase. J Biol Chem. 1983 Sep 25;258(18):11174–11184. [PubMed] [Google Scholar]
  12. Mark D. F., Richardson C. C. Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1976 Mar;73(3):780–784. doi: 10.1073/pnas.73.3.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mills D. R., Kramer F. R. Structure-independent nucleotide sequence analysis. Proc Natl Acad Sci U S A. 1979 May;76(5):2232–2235. doi: 10.1073/pnas.76.5.2232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mizusawa S., Nishimura S., Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Modrich P., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits. J Biol Chem. 1975 Jul 25;250(14):5515–5522. [PubMed] [Google Scholar]
  16. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smith L. M., Sanders J. Z., Kaiser R. J., Hughes P., Dodd C., Connell C. R., Heiner C., Kent S. B., Hood L. E. Fluorescence detection in automated DNA sequence analysis. Nature. 1986 Jun 12;321(6071):674–679. doi: 10.1038/321674a0. [DOI] [PubMed] [Google Scholar]
  19. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES