General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures.
(13)C, (15)N 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.
René
Brigitte
B
Département de Biologie et Pharmacologie Structurales, UMR 8113 CNRS - LBPA Ecole Normale Supérieure de Cachan, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805, Villejuif Cedex, France.
Masliah
Grégoire
G
Zargarian
Loussiné
L
Mauffret
Olivier
O
Fermandjian
Serge
S
D002247:Carbon Isotopes; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D008722:Methods; D009587:Nitrogen Isotopes; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D016133:Polymerase Chain Reaction
D002247:Carbon Isotopes; D017931:DNA Primers; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D004247:DNA