PHOTOCLEAVABLE TAGS FOR MANIPULATION OF DNA/RNA

该项目的目标是开发一种新的隔离技术 以及基于光裂解技术的DNA/RNA检测 寡核苷酸标签(PC-标签)。在第一阶段，开发了新的方法 使用PC-亚磷酰胺将PC-标签引入到寡核苷酸上 和PC-核苷酸。其中几种试剂是合成的，它们 促进将PC标签引入到5‘端的 寡核苷酸在其自动固体载体合成中的化学作用 (PC-亚磷酰胺)或在体外反应中酶促(PC- 核苷酸)。PC-寡核苷酸可以固定在固体上 用半抗原、荧光团或其他物质支撑或特别标记 可检测的组。然后，可以将寡核苷酸从 支撑物或标签通过短暂暴露在紫外光下而移除。在阶段中 这项新技术将通过i)开发一种通用的 合成和整合各种不同的 DNA/RNA中的PC标签；II)评价DNA/RNA修饰后的性质 利用使用包括高效液相色谱在内的几种分析技术的各种PC标签， 表面等离子体共振(SPR)和质谱学；以及iii) 开发和测试该PC标签的各种有前景的应用 技术包括自动分离聚合酶链式反应产物、多个 DNA/RNA的探测和基于MALDI-MS的快速DNA测序和DNA 诊断。 建议的商业应用： 可光切割的寡核苷酸将在 涉及分离和检测DNA/RNA的技术。产品 包括可光裂解亚磷酰胺和可光裂解核苷酸 检测和分离DNA/RNA的试剂盒.自动 DNA/RNA的操作系统和快速DNA的开发 序列分析和基于光切割的DNA诊断 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.