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Improved DNA Polymerases for Genomic Sequencing

用于基因组测序的改进 DNA 聚合酶

基本信息

批准号：
7405421
负责人：
Thomas William Schoenfeld
金额：
$ 33.23万
依托单位：
LUCIGEN CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-27 至 2009-03-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：本项目提出的是，现有DNA聚合酶的不足增加了成本，限制了DNA测序的速度，而这里开发的嗜热噬菌体DNA聚合酶将大大提高自动化荧光测序，以及正在开发的新技术。噬菌体（tm） DNA聚合酶极大地扩展了可用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dna的早期发现。数百个新的噬菌体dna基因已被发现，其中10个已被表达。其中第一个已经证明了几个特征，应该显著提高测序敏感性，减少空白，减少错误和简化模板制备。这些包括通过富含GC的模板高效合成，至少两类链终止子的高效结合，荧光标记核苷酸的结合，对有问题的二核苷酸的碱基调用的改进，高效率的合成，高效的缺口起始，以及使用RNA模板的能力。基于噬菌体dna独特的生化属性组合，我们还提出了Sanger测序的新方法，以进一步提高通量和灵敏度。我们预计第二阶段将导致一种PyroPhage DNAP作为下一代自动荧光测序酶的开发，以及三种或更多的PyroPhage DNAP作为各种其他平台的改进测序试剂。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破译基因组序列方法的可靠性。多年来，人们已经知道，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，无法在标准反应的高温下存活下来，也没有办法分离出合适的酶。使用一种新方法，该项目已经发现了数百种这样的酶，并生产了10种。其中一种已被详细研究，正如预期的那样，它具有有望改善测序的特征。