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Journal of Biomolecular NMR

, Volume 36, Issue 3, pp 137–146 | Cite as

General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

  • Brigitte René
  • Grégoire Masliah
  • Loussiné Zargarian
  • Olivier Mauffret
  • Serge FermandjianEmail author
Article

Summary

13C, 15N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments.

Keywords

DNA isotope labeling NMR PCR structural analysis 

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Notes

Acknowledgement

This work was financially supported by a grant from the French Ministry of Research (to G.M.).

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Brigitte René
    • 1
  • Grégoire Masliah
    • 1
  • Loussiné Zargarian
    • 1
  • Olivier Mauffret
    • 1
  • Serge Fermandjian
    • 1
    Email author
  1. 1.Département de Biologie et Pharmacologie StructuralesUMR 8113 CNRS – LBPA Ecole Normale Supérieure de Cachan, Institut Gustave RoussyVillejuif CedexFrance

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