A PCR-based method for uniform 13C/15N labeling of long DNA oligomers.

Abstract

A polymerase chain reaction (PCR)-based method is described for uniform 13C/15N labeling of DNA duplexes. In this method, multiple copies of a blunt-ended duplex are cloned into a plasmid with each copy containing the sequence of interest and the restriction HincII sequences at the 5' and 3' ends. PCR with uniformly 13C/15N-labeled dNTP precursors results in a labeled DNA duplex containing multiple copies of the sequence of interest. Use of bi-directional primers, instead of self-priming [Louis et al. (1998) J. Biol. Chem. 273, 2374-2378], produces a DNA fragment of unique length. Twenty-four cycles of PCR of this purified product followed by restriction and purification gives (with 30% yield) the uniformly 13C/15N-labeled duplex sequence for multi-nuclear magnetic resonance spectroscopy.

Cite this paper

@article{Chen1998APM, title={A PCR-based method for uniform 13C/15N labeling of long DNA oligomers.}, author={X. Y. Chen and Subramanian Mariappan and Janet J Kelley and John H. Bushweller and E. Morton Bradbury and Goldi Gupta}, journal={FEBS letters}, year={1998}, volume={436 3}, pages={372-6} }