Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynudeotidyl transferase (original) (raw)

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Section of Biochemistry, Molecular and Cell Biology, Cornell University

Ithaca NY 14853, USA

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Section of Biochemistry, Molecular and Cell Biology, Cornell University

Ithaca NY 14853, USA

*

Present address: Department of Chemistry, University of New Brunswick, Fredericton, NB. Canada

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Section of Biochemistry, Molecular and Cell Biology, Cornell University

Ithaca NY 14853, USA

* To whom correspondence should be sent

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*

Present address: Department of Chemistry, University of New Brunswick, Fredericton, NB. Canada

Author Notes

Received:

23 October 1975

Published:

01 January 1976

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Ranajit Roychoudhury, Ernest Jay, Ray Wu, Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynudeotidyl transferase, Nucleic Acids Research, Volume 3, Issue 1, 1 January 1976, Pages 101–116, https://doi.org/10.1093/nar/3.1.101
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Summary

Terminal deoxynudeotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg +2 ion is replaced by Co ion. The priming efficiency in the presence of Co +2 ion with respect to initial rate tested with 2 single-stranded primer, is 5–6 fold higher than that observed with Mg +2 ion. In the presence of Co +2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3′-ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co +2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3′-terminus of double-stranded DNA. Therefore, without prior treatment with λ exonuclease to expose the 3′ terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3′-ends of all types of DNA molecules for the purpose of invitro construction of recombinant DNA.

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Author notes

*

Present address: Department of Chemistry, University of New Brunswick, Fredericton, NB. Canada

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