The goal of this project is to develop improved methods for the synthesis, isolation anddetection of DNA/RNA based on the development of photocleavable oligonucleotide tags (PC-oligotags). PC-oligotags can be introduced into DNA/RNA chen~iicalfy during its solid-support synthesis by means of PC-phosphoramidites or enzymaticaffy using PC-nucleotides. The PC-oligonucleotides which result can be usefbl in a wide variety of applications in molecular biology including PCR-based site-directed mutagenesis, and DNA/RNA hybridization. In initial work, we have demonstrated that oligonucleotides can be 5'-labeled during their automated synthesis with PC-biotin phosphoramidite; selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin and rapidly released by fight in a phosphorylated form for subsequent use in recombinant DNA methods. During Phase 1, we plan to synthesize and evaluate several promising PC-phosphoramidite and PC-nucleotide compounds. Improved methodology in molecular biology based on PC-oligotags will be evaluated including purification of full-length sequences from both synthetically produced DNA/RNA and PCR-based site-directed mutagenesis. During Phase II, PC-oligotags will be used for the automation of the synthesis, isolation and detection of oligonucleotides and will also be applied to specific problems in genetic and developmental biology.
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