Evolving Novel Polymerases for Genome Sequencing

描述(由申请人提供)：有很大的动力来开发低成本的基因组测序技术。虽然传统测序方法已经取得了重大进展，但为了减少成本和所需时间，使基因组测序变得更加可行，需要开发新的方法。事实上，许多新的和有希望的方法正在开发中。然而，这些新的方法通常受到DNA聚合酶不能用修饰的脱氧核苷酸三磷酸(DNTPs)合成DNA的限制。我们建议使用我们最近开发的基于活性的选择系统来进化DNA聚合酶，从而有效地识别这些修饰的dNTPs，从而克服发展新的测序方法的最重要的障碍之一。该选择系统是基于DNA聚合酶文库及其DNA底物在噬菌体上的共展示，使得识别修饰的dNTPs的突变体共价连接生物素-dUTP，从而允许它们的选择性分离。除了选择外，还提出了各种现代基因改组方法来产生聚合酶多样性。该提案的具体目标是：具体目标1：进化利用荧光团标记的dNTPs有效合成DNA的DNA聚合酶，以实现新的测序合成方法。具体目标2：发展能有效整合可逆性荧光团标记的带有3‘-O-烯丙基保护基团的dNTPs的聚合酶，然后在双重去保护后继续合成。具体目标3：在实际测序应用中协作评估进化聚合酶。更适合于测序应用的聚合酶的进化，以及它们在实际测序方法中的评估，有望极大地促进方法的发展，并有助于使基因组测序变得更加常见。

DESCRIPTION (provided by applicant): There is great incentive for developing low cost technologies for sequencing genomes. While significant advances have been made in conventional sequencing methods, the reductions in cost and time required to make genome sequencing more feasable require the development of new methodologies. Indeed many new and promising methods are under development. However, these new approaches are generally limited by the inability of DNA polymerases to synthesize DNA with modified deoxynucleotide triphosphates (dNTPs). We propose to use our recently developed activity-based selection system to evolve DNA polymerases that efficiently recognize these modified dNTPs, and thus overcome one of the most significant barriers to the development of the novel sequencing methods. The selection system is based on the co-display on phage of DNA polymerase libraries and their DNA substrates, such that mutants that recognize the modified dNTPs covalently attach a biotin-dUTP, allowing for their selective isolation. In addition to the selections, various modern gene shuffling approaches are proposed to generate polymerase diversity. The Specific Aims of the proposal are: Specific Aim 1: Evolve DNA polymerases that efficiently synthesize DNA using fluorophore-labeled dNTPs to enable novel synthesis-by-sequencing methodologies. Specific Aim 2: Evolve polymerases that efficiently incorporate reversibly fluorophore tagged dNTPs with 3'-O-allyl protecting groups and then continue synthesis after double deprotection. Specific Aim 3: Collaborate to evaluate evolved polymerases in actual sequencing applications. The evolution of polymerases better suited for sequencing applications, and their evaluation in the actual sequencing methodologies, promises to dramatically facilitate the development of the methods and help make genome sequencing more commonplace.