Advertisement

Restriction Analysis of Cloned Heteropolymeric DNA Synthesized in vitro by TdT

  • G. Damiani
  • E. Palla
  • V. Sgaramella
  • A. I. Scovassi
  • U. Bertazzoni
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 145)

Abstract

It is known that the enzyme terminal deoxynucleotidyl transferase (TdT) catalyses in vitro the polymerization of the four deoxynucleosides triphosphates in a sequence which can be considered largely random. The resulting copolymers can be varied in their composition e.g. by changes in the kind of divalent cation used in the reaction (1). The only experimental data available on the sequence derive from the incorporation of the four precursors and from the enzymatic degradation and nearest neighbor analysis of the product (2,3). None of these approaches yields direct insights on the actual sequence of the polymeric chains synthesized in vitro by TdT.

Keywords

Terminal Deoxynucleotidyl Trans Deoxynucleosides Triphosphate Neighbor Analysis Enzyme Terminal Deoxynucleotidyl Transferase Restriction Site Mapping 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    K. Kato, J. M. Goncalves, G. E. Houts and F.J. Bollum, Deoxynucleotide-polymerizing enzymes of calf thymus gland. II Properties of the terminal deoxynucleotidyl transferase, J. Biol. Chem. 242: 2780 (1967).PubMedGoogle Scholar
  2. 2.
    F. J. Bollum, Preparation of oligodeoxynucleotides, in “Procedures in Nucleic Acid Research” (G. Cantoni and D. Davies,eds) pp. 592–599, Harper, New York (1966).Google Scholar
  3. 3.
    R. L. Ratliff, A. W. Schwartz, V. N. Kerr, D. L. Williams, D.G. Ott and F.N. Hayes, Heteropolydeoxynucleotides synthesized with Terminal Deoxynucleotidyl Transferase. II Nearest neighbor frequencies and extent of digestion by micrococcal Deoxyribonuclease. Biochemistry, 7:412 (1963).CrossRefGoogle Scholar
  4. 4.
    S. D. Ehrlich, DNA cloning in Bacillus subtilis. Proc. Natl. Acad. Sci. USA, 75: 1433(1978)Google Scholar
  5. 5.
    L. Villa-Komaroff, A. Esfradiatis, S. Broome, P. Lomedico, R. Tizard, S. Naber, W. L. Chick and W. Gilbert, A bacterial clone synthetizing proinsulin, Proc. Natl. Acad. Sci. USA 75:3727(1978).Google Scholar
  6. 6.
    R. C. A. Yang, J. Lis and R. Wu, Elution of DNA from agarose gels after electrophoresis, in “Methods in Enzymology”, R. Wu, ed., vol. 68, p. 176, Academic Press, (1979).Google Scholar
  7. 7.
    C. Winberg and M. L. Hammarskjold, Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA, Nucleic Ac. Res., 8:253 (1980).CrossRefGoogle Scholar
  8. 8.
    A. M. Maxam and W. Gilbert, Sequencing end-labeled DNA with basespecific chemical cleavage, in “Methods in Enzymology VL. Grossman and K. Moldave,eds, vol. 65, p. 499, Academic press, (1980).Google Scholar
  9. 9.
    C. P. D. Tu and S. N. Cohen, 3’-end labeling of DNA with 32P cordycepin-S’-triphosphate. Gene 10: 177 (1980).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1982

Authors and Affiliations

  • G. Damiani
    • 1
  • E. Palla
    • 1
  • V. Sgaramella
    • 1
  • A. I. Scovassi
    • 1
  • U. Bertazzoni
    • 1
  1. 1.Istituto CNR Genetica Biochimica ed EvoluzionisticaPaviaItaly

Personalised recommendations