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An Integrated System for DNA Sequencing by Synthesis

DNA 合成测序集成系统

基本信息

批准号：
7923565
负责人：
JINGYUE JU
金额：
$ 40.48万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-11 至 2010-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The objective of the proposed research is to develop an integrated system for DNA sequencing by synthesis (SBS) using cleavable fluorescent nucleotide reversible terminators. We will (1) design and synthesize a library of 2'-deoxynucleotide analogues that each consists of a unique fluorescent dye attached to the base through a cleavable linker, and whose 3'-OH group is modulated as a reversible terminator in polymerase reaction. These nucleotide analogues are designed in such a way that after they are incorporated into the growing strand of DNA in the polymerase reaction, the fluorescent dyes are cleaved by irradiation at 355 nm or by specific chemical reagents in a mild condition that is compatible with DNA, and the 3'-OH can be regenerated to continue the polymerase reaction; (2) develop an "ePCR-bead-on-chip (ePBC)" approach for amplifying and immobilizing DNA products for sequencing by synthesis (SBS). The ePBC approach is based on emulsion PCR on beads and then covalently immobilizing the beads, each containing many copies of unique amplified DNA templates on a chip; (3) evaluate and implement a 4-color SBS system consisting of a laser induced photolysis device, a liquid handling module, and a high sensitivity CCD based 4-color fluorescence imager for sequencing a variety of DNA templates; (4) develop a library construction method for pair-end sequencing of DNA clones using the sequencing chemistry developed in aim 1, the ePBC DNA chip that is developed in aim 2, and the 4-color SBS system in aim 3 for sequencing BAC clones from bacteria and other model organisms. This new system for genome sequencing and resequencing will have wide applications in biology and biomedical research.

描述（由申请人提供）：拟定研究的目的是开发一种使用可裂解荧光核苷酸可逆终止子的DNA合成测序（SBS）集成系统。我们将（1）设计和合成一个2 '-脱氧核苷酸类似物库，每个类似物都由通过可切割接头连接到碱基上的独特荧光染料组成，并且其3'-OH基团被调节为聚合酶反应中的可逆终止子。这些核苷酸类似物以这样的方式设计，即在聚合酶反应中将它们掺入到DNA的生长链中之后，荧光染料通过在355 nm处照射或通过与DNA相容的温和条件下的特定化学试剂裂解，并且3 ′-OH可以再生以继续聚合酶反应;（2）开发用于扩增和固定DNA产物以用于合成测序（SBS）的“ePCR-bead-on-chip（ePBC）”方法。ePBC方法基于在珠上的乳液PCR，然后共价固定珠，每个珠在芯片上含有许多拷贝的独特扩增的DNA模板;（3）评估和实施由激光诱导光解装置、液体处理模块和基于高灵敏度CCD的四色荧光成像仪组成的四色SBS系统，用于对各种DNA模板进行测序;（4）利用目的1中开发的测序化学、目的2中开发的ePBC DNA芯片和目的3中的四色SBS系统开发用于DNA克隆的双末端测序的文库构建方法，用于对来自细菌和其他模式生物的BAC克隆进行测序。这种新的基因组测序和重测序系统将在生物学和生物医学研究中有广泛的应用。