A PCR-BASED METHOD FOR UNIFORM C-13 N-15 LABELING OF LONG DNA OLIGOMERS/

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