Improved DNA Polymerases for Genomic Sequencing

描述(由申请人提供)：该项目提出，现有DNA聚合酶的不足增加了成本并限制了DNA测序的速度，这里开发的嗜热噬菌体DNA聚合酶将极大地改进自动荧光测序，以及正在开发的新技术。PyroPhage(Tm)DNA聚合酶极大地扩展了现有聚合酶的分子多样性和生化能力。对传统微生物学方法的依赖阻碍了这些噬菌体DNAP的早期发现。已经发现了数百个新的火噬菌体DNAP基因，其中10个已经表达。其中第一个已经证明了几个特征，这些特征应该显著提高测序灵敏度、减少差距、减少错误和简化模板准备。这些包括通过富含GC的模板进行高效合成、高效地掺入至少两类链终止子、掺入荧光标记的核苷酸、改进有问题的二核苷酸的碱基调用、合成的效率高、在缺口处有效地引发以及使用RNA模板的能力。基于火噬菌体DNAP独特的生化属性组合，我们还建议开发新的Sanger测序方法，以进一步提高吞吐量和灵敏度。我们预计，第二阶段将导致开发一种用于自动化荧光测序的下一代酶，以及三种或更多用于各种其他平台的改进测序试剂的火噬菌体DNAP。该项目的目标是通过提供一种在测序反应中表现更好的新型酶来降低成本并提高基因组序列破译方法的可靠性。多年来，人们一直知道感染微生物(噬菌体)的酶衍生病毒有可能降低测序成本，但现有的噬菌体酶都不够稳定，不足以在标准反应的高温下生存，也不存在分离适当酶的手段。使用一种新的方法，该项目已经发现了数百种这样的酶，并产生了10种。其中一个已经被详细研究过，正如预期的那样，它具有有望改善测序的特征。

DESCRIPTION (provided by applicant): This project proposes is that deficiencies in available DNA polymerases increase costs and limit the rate of DNA sequencing and that thermophilic phage DNA polymerases developed here will substantially improve automated fluorescent sequencing, as well as newer technologies being developed. PyroPhage(tm) DNA Polymerases greatly expand the molecular diversity and biochemical capabilities of available polymerases. Reliance on traditional microbiology approaches has prevented earlier discovery of these phage DNAPs. Hundreds of new PyroPhage DNAP genes have been discovered, ten of which have been expressed. The first of these has demonstrated several characteristics that should significantly improve sequencing sensitivity, reduce gaps, reduce errors and simplify template preparation. These including efficient synthesis through GC rich templates, efficient incorporation of at least two classes of chain terminators, incorporation of fluorescently labeled nucleotides, improved base calling of problematic dinucleotides, high efficiency of synthesis, efficient initiation at nicks, and the ability to use an RNA template. Based on a unique combination of biochemical attributes of the PyroPhage DNAP, we also propose the development of new approaches to Sanger sequencing to further improve throughput and sensitivity. We anticipate that Phase II will result in the development of one PyroPhage DNAP as the next generation enzyme for automate fluorescent sequencing, and three or more PyroPhage DNAPs as improved sequencing reagents for a variety of other platforms. The goal of this project is to reduce the cost and improve the reliability of methods for deciphering genomic sequences by providing a new class of enzymes that perform better in the sequencing reaction. It has been known for years that enzymes derived viruses that infect microbes (phage) have potential to reduce sequencing costs but none of the existing phage enzymes was stable enough to survive the high temperatures of the standard reaction and no means of isolating an appropriate enzyme existed. Using a new approach, this project has discovered hundreds of such enzymes and produced ten. One has been studied in detail and, as expected, it has characteristics that are expected to improve sequencing.