High Resolution Fluorescent Melting Curve Analysis

简介(申请者摘要)：我们的长期目标是 用于快速、自动和灵敏变异的仪器和技术 核酸的分析。LightCykerTM一直是我们的同质化 系统的基因分型平台。包括荧光杂交探针 在反应开始时，它们被用于实时PCR监测和 基因分型。在快速循环PCR之后，缓慢加热反应(0.1度 C/S)和产品通过独特的探头/目标熔化温度进行识别。 整个分析是在20分钟内从基因组DNA中完成的，并且没有任何 样本操作。荧光熔融曲线法是一种实用而流行的分析方法 已建立的突变/多态的重复基因分型方法。在……里面 第一阶段，我们建造并测试了一台新的“高分辨率”熔化仪器。 曲线分析。高分辨率熔化仪器的分辨率降低了十分之一 测量误差超过LightCykerTM，提供了更高的灵敏度 突变检测及其在突变扫描中的应用。在第二阶段，我们计划 将高分辨率仪器的“低噪音”组件集成到 修改后的LightCykerTM可实现快速放大和平滑融化 曲线基因分型。数学软件算法将为 敏感的变异检测和标记未知的突变序列。该实用程序 新的仪器和算法的性能将由同质的 扫描p53基因关键外显子内的突变。 建议的商业应用： 我们建议开发仪器和分析软件，使同质 首次进行突变扫描。该系统将用于扫描P53肿瘤 抑制基因，该基因在几乎所有癌症侵袭性生长时都发生了突变。 该仪器和化验将可用于商业上的继续研究 以期为肿瘤的预后和治疗提供临床检测依据。这个 一般的方法也可以应用于其他基因。

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.