An Integrated System for DNA Sequencing by Synthesis

描述（由申请人提供）：拟议研究的目的是开发一个使用可切割荧光核苷酸可逆终止体的DNA合成测序（SBS）集成系统。我们将(1)设计并合成一个2'-脱氧核苷酸类似物库，每个类似物由一种独特的荧光染料组成，通过可切割的连接物连接到碱基上，其3'-OH基团在聚合酶反应中作为可逆终止物进行调节。这些核苷酸类似物被设计成在聚合酶反应中与正在生长的DNA链结合后，在355nm照射下或在与DNA兼容的温和条件下用特定的化学试剂切割荧光染料，3'-OH可以再生以继续聚合酶反应；(2)开发一种“ePCR-bead-on-chip （ePBC）”方法，用于扩增和固定DNA产物，用于合成测序（SBS）。ePBC方法是基于乳剂PCR珠，然后共价固定珠，每个珠包含许多拷贝独特的扩增DNA模板在芯片上；(3)评估并实现由激光诱导光解装置、液体处理模块和基于CCD的高灵敏度4色荧光成像仪组成的4色SBS系统，用于多种DNA模板的测序；(4)利用aim 1开发的测序化学、aim 2开发的ePBC DNA芯片和aim 3开发的4色SBS系统对细菌和其他模式生物的BAC克隆进行测序，建立DNA克隆对端测序的文库构建方法。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。

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.