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. 1979 Apr;6(4):1323–1333. doi: 10.1093/nar/6.4.1323

Influence of nucleotide sequence adjacent to duplex DNA termini on 3' terminal labeling by terminal transferase.

R Roychoudhury, C P Tu, R Wu
PMCID: PMC327775  PMID: 450697

Abstract

We have analyzed the effect of base-pairing at the exposed ends of Hinc II fragments of SV40 DNA on the efficiency of ribonucleotide incorporation catalyzed by terminal transferase. Wide variations in the labeling efficiency of individual DNA fragments have been observed. To elucidate the nature of this variation at the molecular level, we have correlated this effect with nucleotide sequence adjacent to the cleavage site. We found that a G:C base pair right at the exposed end drastically reduces the incorporation of ribonucleotides. Furthermore, the higher the number of G:C base pairs adjacent to the exposed end, the greater the reduction in labeling efficiency. From these results, we conclude that the labeling efficiency is determined by the degree of 'terminal breathing' of the DNA molecule at the exposed end of the duplex.

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Selected References

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

  1. BOLLUM F. J. Thermal conversion of nonpriming deoxyribonucleic acid to primer. J Biol Chem. 1959 Oct;234:2733–2734. [PubMed] [Google Scholar]
  2. Brownlee G. G., Sanger F. Chromatography of 32P-labelled oligonucleotides on thin layers of DEAE-cellulose. Eur J Biochem. 1969 Dec;11(2):395–399. doi: 10.1111/j.1432-1033.1969.tb00786.x. [DOI] [PubMed] [Google Scholar]
  3. Fiers W., Contreras R., Haegemann G., Rogiers R., Van de Voorde A., Van Heuverswyn H., Van Herreweghe J., Volckaert G., Ysebaert M. Complete nucleotide sequence of SV40 DNA. Nature. 1978 May 11;273(5658):113–120. doi: 10.1038/273113a0. [DOI] [PubMed] [Google Scholar]
  4. Jay E., Bambara R., Padmanabhan R., Wu R. DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping. Nucleic Acids Res. 1974 Mar;1(3):331–353. doi: 10.1093/nar/1.3.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jensen R. H., Wodzinski R. J., Rogoff M. H. Enzymatic addition of cohesive ends to T7 DNA. Biochem Biophys Res Commun. 1971 Apr 16;43(2):384–392. doi: 10.1016/0006-291x(71)90765-0. [DOI] [PubMed] [Google Scholar]
  6. Kössel H., Roychoudhury R., Fischer D., Otto A. 3' End-group labeling and partial sequence determination of oligodeoxynucleotides. Methods Enzymol. 1974;29:322–341. doi: 10.1016/0076-6879(74)29030-x. [DOI] [PubMed] [Google Scholar]
  7. Kössel H., Roychoudhury R. Synthetic polynucleotides. Ther terminal addition of riboadenylic acid to deoxyoligonucleotides by terminal deoxynucleotidyl transferase as a tool for the specific labelling of deoxyoligonucleotides at the 3'-ends. Eur J Biochem. 1971 Sep 24;22(2):271–276. doi: 10.1111/j.1432-1033.1971.tb01541.x. [DOI] [PubMed] [Google Scholar]
  8. Lobban P. E., Kaiser A. D. Enzymatic end-to end joining of DNA molecules. J Mol Biol. 1973 Aug 15;78(3):453–471. doi: 10.1016/0022-2836(73)90468-3. [DOI] [PubMed] [Google Scholar]
  9. Reddy V. B., Thimmappaya B., Dhar R., Subramanian K. N., Zain B. S., Pan J., Ghosh P. K., Celma M. L., Weissman S. M. The genome of simian virus 40. Science. 1978 May 5;200(4341):494–502. doi: 10.1126/science.205947. [DOI] [PubMed] [Google Scholar]
  10. Roychoudhury R., Jay E., Wu R. Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acids Res. 1976 Apr;3(4):863–877. doi: 10.1093/nar/3.4.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Roychoudhury R., Kössel H. Synthetic polynucleotides. Enzymic synthesis of ribonucleotide terminated oligodeoxynucleotides and their use as primers for the enzymic synthesis of polydeoxynucleotides. Eur J Biochem. 1971 Oct 14;22(3):310–320. doi: 10.1111/j.1432-1033.1971.tb01546.x. [DOI] [PubMed] [Google Scholar]
  12. Tu C. P., Jay E., Bahl C. P., Wu R. A reliable mapping method for sequence determination of oligodeoxyribonucleotides by mobility shift analysis. Anal Biochem. 1976 Jul;74(1):73–93. doi: 10.1016/0003-2697(76)90311-0. [DOI] [PubMed] [Google Scholar]
  13. Wu R., Taylor E. Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA. J Mol Biol. 1971 May 14;57(3):491–511. doi: 10.1016/0022-2836(71)90105-7. [DOI] [PubMed] [Google Scholar]

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