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

描述(申请人提供)：在人类基因组完整序列的基础上，目前正在加紧努力，通过单核苷酸多态(SNP)作图或直接关联来确定常见疾病的潜在遗传联系。以快速、高通量和低成本为重点的SNP检测技术的发展将是遗传信息在应用医学中应用的重大进步。R41提案的目的是开发碱基加成测序方案(BASS)，潜在地应用于从基因组DNA材料中扩增的PCR扩增的靶向微型测序。我们已经证明了BASS的可行性，并确定了DNA测序应用程序开发中的主要挑战。也就是说，它们是(A)不同的2-硝基苯基核酸三磷酸盐的不均匀的紫外线去保护和(B)商业上可获得的DNA聚合酶对3‘-(取代-2-硝基)-dNTPs的低掺入效率。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，它对四个碱基显示了一致的去保护效率，并通过随机突变筛选鉴定了新的Taq DNA聚合酶突变体。BASS技术的可行性测试将是在所有16个2碱基延伸组合中进行两个周期的逐步DNA合成(即两轮结合和去保护)。这项研究的结果将提供对循环效率的更准确的估计，这将是序列读取长度的指标。这些目标的成功实施将展示BASS技术在从头发现SNP方面的应用。

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.