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A PCR‐based method for uniform 13 C/ 15 N labeling of long DNA oligomers
Author(s) -
Chen Xian,
Santhana Mariappan S.V.,
Kelley John J.,
Bushweller John H.,
Bradbury E.Morton,
Gupta Goutam
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01162-4
Subject(s) - duplex (building) , dna , microbiology and biotechnology , sequence (biology) , sticky and blunt ends , polymerase chain reaction , chemistry , biology , restriction enzyme , gene , genetics
A polymerase chain reaction (PCR)‐based method is described for uniform 13 C/ 15 N 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 Hin cII sequences at the 5′ and 3′ ends. PCR with uniformly 13 C/ 15 N‐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 13 C/ 15 N‐labeled duplex sequence for multi‐nuclear magnetic resonance spectroscopy.