Cyclic enzymatic solid phase synthesis of isotopically labeled DNA oligonucleotides.
Isotopic labeling of DNA using standard solid phase synthesis requires expensive phosphoramidites 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 MgCl2/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.
Khan
Ahmed M
AM
Departments of Chemistry and Biology, Georgia State University, Atlanta, Georgia 30303, USA.
Mishra
Subrata H
SH
Germann
Markus W
MW
D001483:Base Sequence; D002247:Carbon Isotopes; D020650:Combinatorial Chemistry Techniques / Q000191:economics; D003500:Cyclization; D004247:DNA / Q000138:chemical synthesis*; D004853:Epoxy Resins / Q000737:chemistry; D015636:Magnesium Chloride / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D009841:Oligonucleotides / Q000138:chemical synthesis*
D002247:Carbon Isotopes; D004853:Epoxy Resins; D009587:Nitrogen Isotopes; D009841:Oligonucleotides; D015636:Magnesium Chloride; D004247:DNA