Gas-phase cleavage and dephosphorylation of universal linker-bound oligodeoxynucleotides.
While base-specific support is commonly used for single-column oligodeoxynucleotide synthesis, the universal linker is critical for high-throughput synthesis of potentially thousands of samples in a single run. Here, we report conditions for cleavage and complete dephosphorylation of two commercial universal linkers, UnySupport and UnyLinker, processed in the gas phase (NH(3)) using our custom device. First, we compared the average yield of T10mers over time (15, 30, 60, 120, and 240 minutes, 40 psi, 80°C and 90°C). For samples processed with water added prior to incubation, we discovered a substantial increase in yield compared to those left dry (up to 55%). This was also the case for samples subjected to increases in chamber pressure (10, 20, 30 and 40 psi, 120 minutes, 80°C and 90°C). Next, we compared the effects of increased temperature, pressure and incubation times on the rates of dephosphorylation. We found the optimum conditions to be either 10 psi, 120 minutes at 80°C or 60 minutes at 90°C; in both cases, water added to columns prior to incubation had a substantial effect on rate of reaction as well as overall yield compared with those left dry. Finally, performance between the two linkers was similar enough to conclude each fulfills the desired requirements for mainstream, high-throughput oligodeoxynucleotide cleavage/deprotection and dephosphorylation in the gas phase.
Stanford Genome Technology Center, Stanford University, 855 S. California Ave, Palo Alto, CA 94304, USA. m.a.jensen@stanford.edu
Michael A
MA
Jensen
Keith M
KM
Anderson
Ronald W
RW
Davis
D005818:Genetic Engineering / Q000295:instrumentation* / Q000379:methods*; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D010766:Phosphorylation; D012263:Ribonucleosides / Q000737:chemistry*
D009838:Oligodeoxyribonucleotides; D012263:Ribonucleosides