Novel Repair Replicase

项目总结 DNA在扩增、文库准备和测序之前和期间的胞嘧啶脱氨基作用是最大的 下一代测序(NGS)数据的误差源(C至A)。聚合酶链式反应过程中的热循环条件 显著加速胞嘧啶脱氨反应。这些错误阻碍了对低丰度变体的检测 作为小型原发和继发肿瘤的驱动突变，以及复杂样本的低保真度，例如 微生物群，对福尔马林固定、石蜡包埋(FFPE)等受损样品尤为严重 组织样本。预计到2025年，仅基于NGS的分子诊断市场就将达到23亿美元， 需要更好的酶。 我们发现了一组新的耐热校对DNA聚合酶A酶与尿嘧啶-DNA 糖基酶(UDG)活性(UDG-DNAP)。初步数据显示，其中一种酶，up19，具有 非常强的3‘外切酶活性，从DNA中去除尿嘧啶的UDG活性，并能够进行强健的PCR 在正常热循环条件下的扩增。在这项提案中，我们将进一步开发这种酶， 商业化一流的、耐热的具有固有UDG活性的DNA聚合酶A的目标 纠正DNA中的尿嘧啶错误。特异性目标1试图在标准聚合酶链式反应中鉴定和优化UP19 对行业标准控制酶的反应。具体目标2旨在测量UP19的保真度 使用Illumina平台和来自快速冷冻肿瘤的高质量DNA对抗行业标准酶 以样本作为模板。具体目标3寻求使用FFPE治疗来测量UP19的尿嘧啶纠错 样本和由此产生的针对工业标准酶和独立中温菌的序列错误率 UDG酶。作为这个项目的结果，我们将对这个新的酶家族有更好的了解，并 更好地理解UP19对NGS应用的适用性。最终目标将是开发出更好的 用于NGS市场的酶，将减少错误率和误诊。 1

PROJECT SUMMARY Cytosine deamination of DNA prior to and during amplification, library preparation, and sequencing is the largest source of errors (C to A) in next-generation sequencing (NGS) data. Thermocycling conditions during PCR sig- nificantly accelerates cytosine deamination. These errors impede the detection of low-abundance variants such as driver mutations for small primary and secondary tumors and lower fidelity for complex samples such as microbiomes and are particularly acute for damaged samples such as formalin-fixed, paraffin-embedded (FFPE) tissue samples. The NGS-based molecular diagnostics market alone is estimated to reach $2.3B by 2025 and needs better enzymes. We have discovered a new group of thermostable proofreading DNA polymerase A enzymes with uracil-DNA glycosylase (UDG) activity (UDG-DNAP). Preliminary data shows that one of these enzymes, UP19, possesses very strong 3’ exonuclease activity, UDG activity for removing uracil from DNA, and is capable of robust PCR amplification under normal thermocycling conditions. In this proposal, we will further develop this enzyme with a goal of commercializing a first-in-class, thermostable proofreading DNA polymerase A with intrinsic UDG activity for correcting uracil mistakes in DNA. Specific Aim 1 seeks to characterize and optimize UP19 in standard PCR reactions against industry standard control enzymes. Specific Aim 2 seeks to measure the fidelity of UP19 against industry standard enzymes using an Illumina platform and high-quality DNA from flash-frozen tumor samples as the template. Specific Aim 3 seeks to measure uracil error correction of UP19 using FFPE treated samples and the resulting sequence error rate against industry standard enzymes and a standalone mesophilic UDG enzyme. As a result of this project, we will have a better understanding of this novel enzyme family and better understand the applicability of UP19 for NGS applications. The ultimate goal will be to develop a better enzyme for the NGS market that will reduce error rates and misdiagnoses. 1