High Resolution Fluorescent Melting Curve Analysis

高分辨率荧光熔解曲线分析

• 批准号: 6337870
• 负责人: PHILIP S BERNARD
• 金额: $ 26.34万
• 依托单位: BIOFIRE DEFENSE, LLC.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6337870
• 关键词: biomedical equipment development; computer program /software; computer system design /evaluation; fluorescence; fluorescence resonance energy transfer; fluorescent dye /probe; nucleic acid denaturation; nucleic acid hybridization; nucleic acid probes; phase change; polymerase chain reaction

DESCRIPTION (Applicant's abstract): Our long-term goal is to advance instrumentation and techniques for rapid, automated and sensitive variant analysis of nucleic acids. The LightCyclerTM has been our homogenous "closed system" platform for genotyping. Fluorescent hybridization probes are included at the beginning of the reaction and are used for realtime PCR monitoring and genotyping. After rapid cycle PCR, the reaction is slowly heated (0.1 degree C/s) and products are identified by unique probe/target melting temperatures. The entire assay is performed from genomic DNA within 20 min and without any sample manipulation. Fluorescent melting curve analysis is a useful and popular method for repetitive genotyping of established mutations/polymorphisms. In phase I, we built and tested a new "high-resolution" instrument for melting curve analysis. The high-resolution melting instrument has 10-fold less measurement error than the LightCyclerTM providing increased sensitivity for mutation detection and utility in mutation scanning. In phase II, we plan to integrate the "low noise" components of the high-resolution instrument into a modified LightCyclerTM to allow both rapid amplification and smooth melting curve genotyping. Mathematical software algorithms will be written for sensitive variant detection and flagging unknown mutant sequences. The utility of the new instrument and algorithms will be demonstrated by homogenously scanning for mutations within critical exons of the p53 gene. PROPOSED COMMERCIAL APPLICATION: We propose developing instrumentation and analysis software that will allow homogenous mutation scanning for the first time. The system will be used for scanning the p53 tumor suppressor gene, which is mutated in almost all cancers at the time of invasive growth. The instrumentation and the assay will be commercially available for continued research on p53 and to provide a clinical test for the prognosis and treatment of cancer. The general method can be applied to other genes, as well.

描述（申请人摘要）：我们的长期目标是推进 用于快速、自动化和灵敏变化的仪器和技术 核酸分析。LightCyclerTM一直是我们同质的“封闭”产品。 系统”平台进行基因分型。包括荧光杂交探针， 在反应开始时，用于实时PCR监测， 基因分型快速循环PCR后，缓慢加热反应物（0.1 C/s）和产物通过独特的探针/靶解链温度鉴定。 整个测定在20分钟内从基因组DNA进行，并且没有任何 样品操作。荧光熔解曲线分析是一种有用的和流行的 对已建立的突变/多态性进行重复基因分型的方法。在 在第一阶段，我们建造并测试了一种新的“高分辨率”熔化仪器， 曲线分析高分辨率的熔化仪器 测量误差比LightCyclerTM提供更高的灵敏度 突变检测和突变扫描中的实用性。在第二阶段，我们计划 将高分辨率仪器的“低噪声”组件集成到 改良LightCyclerTM，可实现快速扩增和平滑解链 曲线基因分型数学软件算法将被编写用于 灵敏的变异检测和标记未知突变序列。本实用 新仪器和算法的性能将通过均匀 扫描p53基因关键外显子内的突变。 拟定商业应用： 我们建议开发仪器和分析软件， 第一次进行突变扫描。 该系统将用于扫描p53肿瘤 抑制基因，这是突变的几乎所有的癌症在侵袭性生长的时候。 仪器和分析将可用于商业研究 并为癌症的预后和治疗提供临床测试。 的 一般的方法也可以应用于其他基因。