CYCLIC ENZYMATIC SOLID PHASE SYNTHESIS OF ISOTOPICALLY LABELED DNA OLIGONUCLEOTIDES

摘要

Isotopic labeling of DNA using standard solid phase synthesis requires expensive phospho-ramidites that are used in large excess. We have developed a protocol where enzymatic, cyclic, solid phase synthesis of DNA facilitates a more economical use of the less expensive labeled DNA triphosphates (dNTP). In this approach, the DNA template is immobilized on an epoxy-activated solid support. Both the support and the linkage between DNA and resin are inert to high pH conditions which are required for product release in this scheme. Efficient covalent attachment of the DNA to the resin was achieved when the reaction was carried out in MgCl_2/CAPS. The enzymatic fill in reaction as well as product release and recycling conditions were optimized for efficient reuse of dNTPs without any purification. The developed protocol was used to generate a selectively [~(13)C, ~(15)N] G labeled 10-mer duplex.