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）：拟议的研究的目的是使用可裂解的荧光核苷酸可逆终端来开发通过合成（SBS）进行DNA测序的集成系统。我们将（1）设计和合成一个由2'-脱氧核苷酸类似物组成的库，每个核苷酸类似物由可裂解的连接器连接到底座上的独特荧光染料，其3'-OH组在聚合酶反应中被调制为可逆的终结剂。这些核苷酸类似物的设计方式是使它们在聚合酶反应中掺入生长的DNA生长链后，荧光染料通过355 nm的照射或在与DNA，轻度兼容的DNA，与DNA相兼容的特定化学试剂中裂解。 3'-OH可以再生以继续聚合酶反应。（2）开发一种“ EPCR珠片（EPBC）”方法，用于扩增和固定通过合成测序（SBS）进行测序的DNA产物。 EPBC方法基于珠子上的乳液PCR，然后共价固定珠子，每个珠子都包含许多芯片上独特的放大DNA模板的副本。（3）评估和实现由激光诱导的光解器，液体处理模块和高灵敏度CCD基于CCD的4色荧光成像仪组成的4色SBS系统，用于对多种DNA模板进行测序；（4）使用AIM 1中开发的测序化学，在AIM 2中开发的EPBC DNA芯片以及AIM 3中的4色SBS System在AIM 3中开发的测序化学方法，开发了用于DNA克隆的成对测序的库构造方法来自细菌和其他模型生物。这种用于基因组测序和重新取证的新系统将在生物学和生物医学研究中广泛应用。