A PCR-based method for uniform 13C/15N labeling of long DNA oligomers.
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.
Chen
X
X
LS-4, LS-8, LS-DO, MS M880, Life Science Division, Los Alamos National Laboratory, NM 87545, USA.
Mariappan
S V
SV
Kelley
J J
JJ
Bushweller
J H
JH
Bradbury
E M
EM
Gupta
G
G
D001483:Base Sequence; D002247:Carbon Isotopes; D004247:DNA / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D004247:DNA