Screening Tag Pol I Variants using 3'-O- Modified-dNTPs

描述（由申请人提供）：基于人类基因组的完整序列，目前正在通过单核苷酸多态性（SNP）作图或直接关联来确定与常见疾病的潜在遗传联系。快速、高通量、低成本的SNP检测技术将是遗传信息应用于应用医学的重大进展。R41提案的目的是开发碱基附加测序方案（BASS），该方案具有从基因组DNA材料中扩增的pcr进行靶向微测序的潜在应用。我们已经证明了BASS的可行性，并确定了DNA测序应用开发中的主要挑战。也就是说，它们是(a)不同的2-硝基苄基核碱基三磷酸不均匀的紫外脱保护和(b) 3'-（取代的2-硝基苄基）- dntps被市购DNA聚合酶结合效率差。为了克服这些障碍，我们提出了“3-0-（取代-2-硝基苄基）- dntps”的合成方法，该方法对4个核碱基具有统一的脱保护效率，并通过随机诱变筛选鉴定出新的Taq DNA聚合酶变体。BASS技术的可行性测试将在所有16个2碱基延伸组合中进行两个周期的逐步DNA合成（即两轮合并和去保护）。本研究的结果将提供一个更准确的循环效率的估计，这将是序列读长的一个指标。这些目标的成功实现将证明BASS技术在新SNP发现中的应用。

DESCRIPTION (provided by applicant): Building upon the complete sequence of the human genome, intense efforts are currently underway to identify the underlying genetic link to common diseases by single nucleotide polymorphism (SNP) mapping or direct association. Technology development focused on rapid, high-throughput, and low cost SNP detection would represent a major advance for the application of genetic information in applied medicine. The aim of the R41 proposal is the development of the Base Addition Sequencing Scheme (BASS) with potential application for targeted mini-sequencing from PCR-amplified from genomic DNA materials. We have demonstrated the feasibility of BASS and have identified the major challenges in development for DNA sequencing applications. Namely, they are (a) non-uniform UV deprotection of the different 2-nitrobenzyl nucleobase triphosphates and (b) poor incorporation efficiencies of 3'-(substituted-2-nitrobenzyl)-dNTPs by commercially available DNA polymerases. To overcome these obstacles, we propose the synthesis of '3-0- (substituted-2-nitrobenzyl)-dNTPs, which show uniform deprotection efficiencies for the four nucleobases and the identification of novel Taq DNA polymerase variants by random mutagenesis screens. The test of feasibility for the BASS technology will be two cycles of stepwise DNA synthesis (that is, two rounds of incorporation and deprotection) in all sixteen 2-base extension combinations. The outcome of this research will provide a more accurate estimate of the cycle efficiency, which will be an indicator of the sequence readlength. Successful implementation of these aims will result would demonstrate the BASS technology for application of de novo SNP discovery.