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. 1985 Nov 11;13(21):7663–7672. doi: 10.1093/nar/13.21.7663

Selective protection of in vitro synthesized cDNA against nucleases by incorporation of phosphorothioate-analogues.

G Schreiber, E M Koch, W J Neubert
PMCID: PMC322078  PMID: 2415922

Abstract

The conditions for the stepwise synthesis of single- (ss) and double-stranded (ds) cDNA using thio-analogues instead of dNTPs are described in this paper. RNA of paramyxovirus Sendai (strain 6/94) serves as template in these experiments. The increased resistance of this alpha S-modified cDNA against several nucleases, like S1-Nuclease, DNase I, Exonuclease III, snake venom Phosphodiesterase (PDE) and the combination of DNase I and PDE is demonstrated.

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Selected References

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

  1. Bartlett P. A., Eckstein F. Stereochemical course of polymerization catalyzed by avian myeloblastosis virus reverse transcriptase. J Biol Chem. 1982 Aug 10;257(15):8879–8884. [PubMed] [Google Scholar]
  2. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  3. Koch E. M., Neubert W. J., Hofschneider P. H. Lifelong persistence of paramyxovirus Sendai-6/94 in C129 mice: detection of a latent viral RNA by hybridization with a cloned genomic cDNA probe. Virology. 1984 Jul 15;136(1):78–88. doi: 10.1016/0042-6822(84)90249-6. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Neubert W. J., Hofschneider P. H., Koprowski H. Search for Sendai 6/94 viral RNA in the antigen-free cell line Cl-C-2 isolated from human multiple sclerosis brain tissue. Infect Immun. 1983 Aug;41(2):675–682. doi: 10.1128/iai.41.2.675-682.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Putney S. D., Benkovic S. J., Schimmel P. R. A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7350–7354. doi: 10.1073/pnas.78.12.7350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. RAZZELL W. E., KHORANA H. G. Studies on polynucleotides. III. Enzymic degradation; substrate specificity and properties of snake venom phosphodiesterase. J Biol Chem. 1959 Aug;234(8):2105–2113. [PubMed] [Google Scholar]
  8. Sippel A. E. Purification and characterization of adenosine triphosphate: ribonucleic acid adenyltransferase from Escherichia coli. Eur J Biochem. 1973 Aug 1;37(1):31–40. doi: 10.1111/j.1432-1033.1973.tb02953.x. [DOI] [PubMed] [Google Scholar]
  9. Vogt V. M. Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Eur J Biochem. 1973 Feb 15;33(1):192–200. doi: 10.1111/j.1432-1033.1973.tb02669.x. [DOI] [PubMed] [Google Scholar]
  10. Vogt V. M. Purification and properties of S1 nuclease from Aspergillus. Methods Enzymol. 1980;65(1):248–255. doi: 10.1016/s0076-6879(80)65034-4. [DOI] [PubMed] [Google Scholar]
  11. Vosberg H. P., Eckstein F. Effect of deoxynucleoside phosphorothioates incorporated in DNA on cleavage by restriction enzymes. J Biol Chem. 1982 Jun 10;257(11):6595–6599. [PubMed] [Google Scholar]
  12. Vosberg H. P., Eckstein F. Incorporation of phosphorothioate groups into fd and phi X174 DNA. Biochemistry. 1977 Aug 9;16(16):3633–3640. doi: 10.1021/bi00635a020. [DOI] [PubMed] [Google Scholar]
  13. Weiss B. Endonuclease II of Escherichia coli is exonuclease III. J Biol Chem. 1976 Apr 10;251(7):1896–1901. [PubMed] [Google Scholar]

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