DEVELOPMENT OF A HOMOGENEOUS GENOTYPING METHOD

单核苷酸取代是最常见的多态性形式 和致病基因突变。的有效方法 对这些突变进行鉴定和分型将在这两个方面都有许多应用 临床诊断和遗传学，如：遗传的产前诊断 疾病，移植前的HLA分型和法医学。的能力 在大群体中键入大量的遗传标记将 加速发现复杂人类疾病中的重要基因， 成人糖尿病、冠状动脉疾病和癌症这 一项提案旨在开发一种高通量、同质的基因分型方法 基于微测序和荧光能量转移。荧光 共振能量转移（FRET）是一种物理现象， 这是一种独特的方法来监测两种荧光染料之间的距离。使用 FRET作为一种报告系统，人们可以监测 微测序基因分型反应中的特异性染料标记核苷酸 而不必分离或纯化DNA引物延伸产物。我们 还计划将PCR和微测序结合起来，以进一步简化 议定书我们将探索用虾碱处理PCR产物 磷酸酶和核酸外切酶I作为去除过量PCR引物的方法 和dNTPs。如果成功，我们将 已经开发了一种单容器，均质基因分型测定，适合于完全 自动化，从而开辟了真正大规模人口的道路 使用遗传标记的研究。

Single nucleotide substitutions are the most frequent form of polymorphism and disease-causing mutations in the human. An efficient method to identify and type these mutations will have many applications in both clinical diagnostics and genetics such as: prenatal diagnosis of genetic diseases, HLA-typing prior to transplantation, and forensics. The ability to type large numbers of genetic markers in large populations will accelerate the discovery of genes important in complex human diseases such as adult-onset diabetes, coronary artery disease, and cancer. This proposal aims to develop a high-throughput, homogeneous genotyping method based on minisequencing and fluorescence energy transfer. Fluorescent resonance energy transfer (FRET) is a physical phenomenon that provides a unique way to monitor the distance between two fluorescent dyes. Using FRET as a reporting system, one can monitor the incorporation of a specific dye-labeled nucleotide in the minisequencing genotyping reaction without having to separate or purify the DNA primer extension products. We also plan to couple PCR and minisequencing to further simplify the protocol. We will explore treatment of PCR products with shrimp alkaline phosphatase and exonuclease I as a way to inactivate excess PCR primers and dNTPs prior to the minisequencing reaction. If successful, we will have developed a one-vessel, homogeneous genotyping assay amenable to full automation, thereby opening up the way to truly large-scale population studies using genetic markers.