Ramanathan Sunita, Rao Basuthkar J, Chary K V R
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai-, 400 005, India.
Biochem Biophys Res Commun. 2002 Jan 25;290(3):928-32. doi: 10.1006/bbrc.2001.6306.
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.
提出了一种用于大规模合成用于核磁共振研究的（13）C和（15）N标记DNA的新方法。在该方法中，一种改良的PCR策略——核酸内切酶敏感重复扩增（ESRA），已被用于扩增目标DNA序列的串联重复序列。模板的设计使得限制性内切酶（RE）位点分隔目标序列的重复序列。然后将ESRA产物克隆到合适的载体中。携带质粒的大肠杆菌细胞在分别以[（13）C]葡萄糖和（15）NH4Cl作为唯一碳源和氮源的基本培养基中生长。通过质粒的RE消化释放目标序列，随后使用PAGE进行纯化。在优化条件下，发现使用该方法制备的（13）C/（15）N标记DNA的产量（约5毫克/升培养物）比其他已知的酶法高出几倍。已使用二维核磁共振技术证实了同位素的成功掺入。
