Skip Navigation



Nucleic Acids Research Advance Access published online on June 24, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp540
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (4944K) Freely available
Right arrow Screen PDF (678K) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
37/17/e114    most recent
gkp540v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Nelissen, F. H. T.
Right arrow Articles by Wijmenga, S. S.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nelissen, F. H. T.
Right arrow Articles by Wijmenga, S. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods online

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

Frank H. T. Nelissen, Frederic C. Girard, Marco Tessari, Hans A. Heus and Sybren S. Wijmenga*

Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, the Netherlands

*To whom correspondence should be addressed. Tel: +31 24 3652678; Fax: +31 24 3652112; Email: s.wijmenga{at}nmr.ru.nl

Received April 7, 2009. Revised June 9, 2009. Accepted June 9, 2009.

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental 13C/15N/2H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2' of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively 13C9/15N3/2H(1',2'',3',4',5',5'')-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively 13C9/15N3/2H(1',2'',3',4',5',5'')-dC and 13C9/15N2/2H(1',2'',3',4',5',5'')-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective 2H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1'–H2' and C2'–H2' resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.