PHOTOCLEAVABLE TAGS FOR MANIPULATION OF DNA/RNA

该项目的目标是开发一种新的隔离技术， 和基于使用光可裂解的DNA/RNA的检测 寡核苷酸标签（PC-标签）。在第一阶段， 用于使用PC-亚磷酰胺将PC-标签引入寡核苷酸 和PC-核苷酸。合成了几种这样的试剂， 促进将PC-标签引入到 寡核苷酸在其自动化固相支持合成过程中的化学反应 （PC-亚磷酰胺）或在体外反应期间酶促反应（PC-亚磷酰胺 核苷酸）。 PC-寡核苷酸可以固定在固体上， 用半抗原、荧光团或其它物质支持或特异性标记 可检测组 然后可以将寡核苷酸从寡核苷酸上分离。 通过短暂暴露于紫外线下去除支撑物或标签。在阶段 II这项新技术将通过i）开发一种通用的 综合和纳入各种不同 ii）评估经修饰的DNA/RNA的性质; 使用多种分析技术包括HPLC， 表面等离子体共振（SPR）和质谱法;以及iii） 开发和测试这种PC标签的各种有前途的应用 技术，包括PCR产物的自动分离， 基于MALDI-MS的快速DNA测序和DNA 诊断 拟定商业应用： 可光裂解的寡核苷酸将具有商业应用， 涉及DNA/RNA分离和检测的技术。产品 包括可光裂解亚磷酰胺和可光裂解的核苷酸 检测和分离DNA/RNA的试剂、试剂盒;自动化 DNA/RNA操作系统;以及快速DNA的开发 序列分析和DNA诊断的基础上使用的光裂解 寡核苷酸和MALDI-MS。

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.