Closed Complex Single Molecule Sequencing

封闭式复杂单分子测序

基本信息

批准号：
7938122
负责人：
John Nelson
金额：
$ 49.5万
依托单位：
GENERAL ELECTRIC GLOBAL RESEARCH CTR
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-28 至 2012-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7938122
关键词：
Active Sites Base Sequence Binding Biological Assay Bread Buffers Characteristics Charge Chemicals Chemistry Clinical Trials Complex Crystallization DNA DNA Binding DNA Polymerase I DNA Primers DNA Sequence DNA-Directed DNA Polymerase Data Analyses Diphosphates Dissociation Divalent Cations Dyes Enzymatic Biochemistry Enzymes Escherichia coli Exploratory/Developmental Grant Family Fluorescence Freezing Genes Genome Genomics Healthcare Human Human Genome Hydroxyl Radical Individual Investigation Ions Knowledge Label Left Length Libraries Lytic Phase Maps Mediating Medical Metals Methods Molecular National Human Genome Research Institute Nucleotides Oligonucleotides Organism Persons Phase Poly I Polymerase Principal Investigator Reaction Reading Reagent Research Resources Savings Scanning Schedule Screening procedure Solid Solutions Speed Surface System Techniques Technology Testing Therapeutic Time Tweens Work analog base chemical reaction conformational conversion cost design divalent metal flexibility gel electrophoresis genetic analysis genome-wide inorganic phosphate instrument instrumentation meetings novel nuclease nucleotide analog polymerization programs research study single molecule trend

项目摘要

DESCRIPTION (provided by applicant): Through almost the entire time that the first Human Genome was being mapped and sequenced, a number of companies (that are now part of GE Healthcare) actively developed and provided essential enzymes, dyes, methods, and instruments to the effort. Now that this effort is complete, NHGRI has recognized that a promising new demand for sequence information will emerge once the cost of obtaining that information is decreased by 1000-fold or more. While a number of promising approaches to collect sequence information quickly and inexpensively are under investigation, most of those approaches involve a complex interplay between dye and nucleotide chemistries, enzymology, instrumentation, and data analysis. GE proposes to use existing enzyme and dye-tagged nucleotide resources in a new way that will simplify the fundamental, front-end chemistry of massively parallel sequencing-by-synthesis. GE's proposed method uses the natural catalytic cycle of DNA polymerase to capture just a single nucleotide on an immobilized primer/template. The nucleotide is captured prior to the chemical reaction step of the polymerization cycle, so it can be tagged with a dye attached to the pyrophosphate, a part that eventually is discarded. However, prior to completing the reaction cycle, the tagged nucleotide is identified using a fluorescence scanner that will scan hundreds of thousands of similar molecules at one time. When scanning is complete, the synthesis cycle is finished by simply adding a buffer containing divalent metal ion. Previous GE research efforts have identified native polymerases that incorporate such tagged nucleotides at least 15- 20% as rapidly as unmodified dNTPs. Thus, this new chemistry will simplify the overall system requirements for sequencing-by-synthesis, permitting much more flexible systems and enabling significantly longer read-lengths.

描述（由申请人提供）：几乎在第一个人类基因组被绘制和测序的整个过程中，许多公司（现在属于 GE Healthcare 的一部分）积极开发并提供了必要的酶、染料、方法和仪器，的努力。现在这项工作已经完成，NHGRI 认识到，一旦获取序列信息的成本降低 1000 倍或更多，就会出现对序列信息有前景的新需求。虽然正在研究许多快速且廉价地收集序列信息的有前途的方法，但大多数这些方法涉及染料和核苷酸化学、酶学、仪器和数据分析之间复杂的相互作用。 GE 提议以一种新的方式使用现有的酶和染料标记的核苷酸资源，这将简化大规模并行合成测序的基本前端化学。 GE 提出的方法利用 DNA 聚合酶的自然催化循环来捕获固定引物/模板上的单个核苷酸。核苷酸在聚合循环的化学反应步骤之前被捕获，因此可以用附着在焦磷酸盐上的染料进行标记，焦磷酸盐是最终被丢弃的部分。然而，在完成反应循环之前，使用荧光扫描仪来识别标记的核苷酸，该扫描仪将一次扫描数十万个相似的分子。扫描完成后，只需添加含有二价金属离子的缓冲液即可完成合成循环。 GE 之前的研究工作已经发现，天然聚合酶掺入此类标记核苷酸的速度至少比未修饰的 dNTP 快 15-20%。因此，这种新的化学方法将简化合成测序的整体系统要求，允许更灵活的系统并实现显着更长的读取长度。