An Integrated System for DNA Sequencing by Synthesis

描述(由申请人提供)：拟议研究的目标是开发一种使用可切割的荧光核苷酸可逆终止子的DNA合成测序(SBS)的集成系统。我们将(1)设计和合成一个2‘-脱氧核苷酸类似物文库，每个文库由一个独特的荧光染料通过一个可切割的连接子连接到碱基上，其3’-OH基团在聚合酶反应中被调节为一个可逆的终止子。这些核苷酸类似物被设计成这样一种方式，当它们在聚合反应中被加入到生长的DNA链中后，荧光染料在355 nm处被辐射或在与DNA相容的温和条件下被特定的化学试剂切割，3‘-OH可以再生以继续聚合反应；(2)发展一种用于扩增和固定DNA产物用于合成测序(SBS)的“EPCRbead-on-Chip(EPBC)”方法。EPBC方法是基于小球上的乳液聚合酶链式反应，然后共价固定小球，每个小球包含多个拷贝的独特的扩增DNA模板在一个芯片上：(3)评估和实现一个四色SBS系统，包括一个激光诱导光解装置，一个液体处理模块，和一个高灵敏度的基于CCD的四色荧光成像仪，用于对各种DNA模板进行测序；(4)利用Aim 1中开发的测序化学、Aim 2中开发的EPBC DNA芯片和Aim 3中的4色SBS系统，建立了DNA克隆双端测序的文库构建方法，用于对细菌和其他模式生物的BAC克隆进行测序。这一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.