A novel approach for uniform (13)C and (15)N labeling of DNA for NMR studies.
A novel method is proposed for large-scale synthesis of (13)C- and (15)N-labeled DNA for NMR studies. In this methodology, endonuclease-sensitive repeat amplification (ESRA), a modified PCR strategy, has been used to amplify tandem repeats of the target DNA sequence. The design of the template is such that restriction enzyme (RE) sites separate repeats of the target sequence. The ESRA product is then cloned into a suitable vector. The Escherichia coli cells harboring the plasmid are grown in minimal medium containing [(13)C]glucose and (15)NH(4)Cl as the sole source of carbon and nitrogen, respectively. The target sequence is released by RE digestion of the plasmid, followed by purification using PAGE. Under optimized conditions, the yield ( approximately 5 mg/liter of culture) of (13)C/(15)N-labeled DNA prepared using this approach is found to be several times higher compared to other known enzymatic methods. Successful incorporation of the isotopes has been confirmed using 2D NMR techniques.
Ramanathan
Sunita
S
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai-, 400 005, India.
Rao
Basuthkar J
BJ
Chary
K V R
KV
D002247:Carbon Isotopes; D004262:DNA Restriction Enzymes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D020080:Tandem Repeat Sequences
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D009838:Oligodeoxyribonucleotides; D004262:DNA Restriction Enzymes