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Nucleic Acids Res. 1994 October 11; 22(20): 4259–4267.
PMCID: PMC331938

Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates.

Abstract

Eight 3'-modified-dNTPs were synthesized and tested in two different DNA template assays for incorporation activity. From this enzymatic screen, two 3'-O-methyl-dNTPs were shown to terminate DNA syntheses mediated by a number of polymerases and may be used as alternative terminators in Sanger sequencing. 3'-O-(2-Nitrobenzyl)-dATP is a UV sensitive nucleotide and was shown to be incorporated by several thermostable DNA polymerases. Base specific termination and efficient photolytic removal of the 3'-protecting group was demonstrated. Following deprotection, DNA synthesis was reinitiated by the incorporation of natural nucleotides into DNA. The identification of this labile terminator and the demonstration of a one cycle stop-start DNA synthesis are initial steps in the development of a novel sequencing strategy.

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Selected References

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  • Hiratsuka T. New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. Biochim Biophys Acta. 1983 Feb 15;742(3):496–508. [PubMed]
  • Ludwig J. A new route to nucleoside 5'-triphosphates. Acta Biochim Biophys Acad Sci Hung. 1981;16(3-4):131–133. [PubMed]
  • Tabor S, Richardson CC. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. [PubMed]
  • Innis MA, Myambo KB, Gelfand DH, Brow MA. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. [PubMed]
  • McClary J, Ye SY, Hong GF, Witney F. Sequencing with the large fragment of DNA polymerase I from Bacillus stearothermophilus. DNA Seq. 1991;1(3):173–180. [PubMed]
  • Kong H, Kucera RB, Jack WE. Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. J Biol Chem. 1993 Jan 25;268(3):1965–1975. [PubMed]
  • Huang P, Farquhar D, Plunkett W. Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases. J Biol Chem. 1990 Jul 15;265(20):11914–11918. [PubMed]
  • Hillebrand GG, McCluskey AH, Abbott KA, Revich GG, Beattie KL. Misincorporation during DNA synthesis, analyzed by gel electrophoresis. Nucleic Acids Res. 1984 Apr 11;12(7):3155–3171. [PMC free article] [PubMed]
  • Skinner JA, Eperon IC. Misincorporation by AMV reverse transcriptase shows strong dependence on the combination of template and substrate nucleotides. Nucleic Acids Res. 1986 Sep 11;14(17):6945–6964. [PMC free article] [PubMed]
  • Yu H, Goodman MF. Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates. J Biol Chem. 1992 May 25;267(15):10888–10896. [PubMed]
  • Goody RS, Müller B, Restle T. Factors contributing to the inhibition of HIV reverse transcriptase by chain-terminating nucleotides in vitro and in vivo. FEBS Lett. 1991 Oct 7;291(1):1–5. [PubMed]
  • Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrman SN, Gallo RC, Bolognesi D, Barry DW, Broder S. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7096–7100. [PubMed]
  • Mitsuya H, Yarchoan R, Broder S. Molecular targets for AIDS therapy. Science. 1990 Sep 28;249(4976):1533–1544. [PubMed]
  • Chidgeavadze ZG, Beabealashvilli RS, Atrazhev AM, Kukhanova MK, Azhayev AV, Krayevsky AA. 2',3'-Dideoxy-3' aminonucleoside 5'-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases. Nucleic Acids Res. 1984 Feb 10;12(3):1671–1686. [PMC free article] [PubMed]
  • Chidgeavadze ZG, Scamrov AV, Beabealashvilli RS, Kvasyuk EI, Zaitseva GV, Mikhailopulo IA, Kowollik G, Langen P. 3'-Fluoro-2',3'-dideoxyribonucleoside 5'-triphosphates: terminators of DNA synthesis. FEBS Lett. 1985 Apr 22;183(2):275–278. [PubMed]
  • Beabealashvilli RS, Scamrov AV, Kutateladze TV, Mazo AM, Krayevsky AA, Kukhanova MK. Nucleoside 5'-triphosphates modified at sugar residues as substrates for calf thymus terminal deoxynucleotidyl transferase and for AMV reverse transcriptase. Biochim Biophys Acta. 1986 Nov 13;868(2-3):136–144. [PubMed]
  • Chidgeavadze ZG, Beabealashvilli R Sh, Krayevsky AA, Kukhanova MK. Nucleoside 5'-triphosphates with modified sugars as substrates for DNA polymerases. Biochim Biophys Acta. 1986 Nov 13;868(2-3):145–152. [PubMed]
  • Kutateladze TV, Kritzyn AM, Florentjev VL, Kavsan VM, Chidgeavadze ZG, Beabealashvilli RS. 3'-Hydroxymethyl 2'-deoxynucleoside 5'-triphosphates are inhibitors highly specific for reverse transcriptase. FEBS Lett. 1986 Oct 27;207(2):205–212. [PubMed]
  • Chinchaladze DZ, Prangishvili DA, Scamrov AV, Beabealashvili RS, Dyatkina NB, Krayevsky AA. Nucleoside 5'-triphosphates modified at sugar residues as substrates for DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochim Biophys Acta. 1989 Jun 1;1008(1):113–115. [PubMed]
  • Krayevsky AA, Tarussova NB, Kuhanova MK, Balzarini J, DeClercq E, Karamov EV, Lukashov VV. Investigation of DNA biosynthesis catalyzed by DNA polymerases: approach of synthesis of compounds with anti-HIV activity. Nucleic Acids Symp Ser. 1991;(24):13–16. [PubMed]
  • Yuzhakov AA, Chidgeavadze ZG, Beabealashvilli R Sh. 3'-Mercapto-2',3'-dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase. FEBS Lett. 1992 Jul 20;306(2-3):185–188. [PubMed]
  • Semizarov DG, Victorova LS, Krayevsky AA, Kukhanova MK. Modified nucleoside 5'-triphosphates containing 2',3'-fused three-membered rings as substrates for different DNA polymerases. FEBS Lett. 1993 Jul 19;327(1):45–48. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Hunkapiller T, Kaiser RJ, Koop BF, Hood L. Large-scale and automated DNA sequence determination. Science. 1991 Oct 4;254(5028):59–67. [PubMed]

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