PHOTOCLEAVABLE TAGS FOR MANIPULATION OF DNA/RNA

点击翻译按钮获取中文摘要

The goal of this project is to develop a new technology for the isolation and detection of DNA/RNA based on the use of photocleavable oligonucleotide tags (PC-tags). During Phase I, new methods were developed for introducing PC-tags onto oligonucleotides using PC-phosphoramidites and PC-nucleotides. Several of these reagents were synthesized which facilitate the introduction of a PC-tag - onto the 5'-end of an oligonucleotide chemically during its automated solid-support synthesis (PC-phosphoramidites) or enzymatically during in vitro reactions (PC- nucleotides). The PC-oligonucleotides can be immobilized on a solid support or specifically labeled with a hapten, fluorophore or other detectable group. The oligonucleotide can then be detached from the support or the label removed by brief exposure to UV-light. During Phase II this new technology will be expanded by i) developing a general strategy for synthesizing and incorporating a wide variety of different PC-tags into DNA/RNA; ii) evaluating the properties of DNA/RNA modified with various PC-tags using several analytical techniques including HPLC, surface plasmon resonance (SPR), and mass spectrometry; and iii) developing and testing a variety of promising applications of this PC-tag technology including the automated isolation of PCR-products, multiple probing of DNA/RNA, and MALDI-MS based fast DNA sequencing and DNA diagnostics. PROPOSED COMMERCIAL APPLICATION: Photocleavable oligonucleotides will have commercial applications in technologies involving the isolation and detection of DNA/RNA. Products include photocleavable phosphoramidite and photocleavable nucleotide reagents, kits for the detection and isolation of DNA/RNA; automated systems for manipulation of DNA/RNA; and the development of fast DNA sequence analysis, and DNA diagnostics based on the use of photocleavable oligonucleotides and MALDI-MS.