René Brigitte, Masliah Grégoire, Zargarian Loussiné, Mauffret Olivier, Fermandjian Serge
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.
J Biomol NMR. 2006 Nov;36(3):137-46. doi: 10.1007/s10858-006-9075-0. Epub 2006 Oct 4.
(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.
生物分子的（13）C、（15）N标记可使核磁共振（NMR）共振峰的归属更容易，并提供更多的NMR参数，这极大地提高了DNA结构的质量。然而，目前还没有像用于蛋白质和RNA那样通用的DNA标记方法。在此，我们描述了一种通用且广泛适用的方法，用于制备可用于NMR研究的同位素标记DNA片段。该方法基于在标记的脱氧核苷酸三磷酸存在下对寡核苷酸进行PCR扩增。由于在要研究的DNA序列的两个重复序列之间插入了短DNA序列（接头），该方法具有很大的灵活性。接头的大小和序列设计为在与目标DNA的连接处产生限制性酶切位点。通过酶切PCR产物可释放出具有所需序列和大小的DNA双链体。通过制备两个与生物学相关的DNA片段验证了该方法的适用性。
