Preparation of uniformly isotope-labeled DNA oligonucleotides for NMR spectroscopy.
Two methods for the large scale preparation of uniformly isotope-labeled DNA for NMR studies have been developed. The first method comprises the growth of a suitable plasmid harboring multiple copies of the desired oligonucleotide in a medium based on 15N and 13C nutrients. The second method uses a polymerase chain reaction (PCR)-based approach with 15N- and/or 13C-labeled deoxynucleoside triphosphates. The novelty of our PCR strategy over existing ones is that the primer and template are the identical molecule, resulting in an exponential growth in the length of the double strand that contains tandem repeats of the target DNA sequence. This novel PCR approach, which we have termed ESRA for endonuclease-sensitive repeat amplification, is easy to use, results in high yields, and can be accomplished at low costs. The utility of both methods is demonstrated for the preparation of a double-stranded 21-mer uniformly labeled with 15N and a double-stranded 17-mer DNA uniformly labeled with 15N and 13C.
Louis
J M
JM
Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Martin
R G
RG
Clore
G M
GM
Gronenborn
A M
AM
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*
D009838:Oligodeoxyribonucleotides; D004247:DNA