A PCR‐based method for uniform 13C/15N labeling of long DNA oligomers
Open Access
- 9 October 1998
- journal article
- Published by Wiley in FEBS Letters
- Vol. 436 (3) , 372-376
- https://doi.org/10.1016/s0014-5793(98)01162-4
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.Keywords
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