Molecular Diagnostic Assays for Genetic Diseases

描述(由申请人提供)：与参考的聚合酶链式反应方法相比，这项建议的第一阶段证明了检测人血液中的因子V莱顿突变和正确确定样本基因的可行性。该协议使用了Lumigen发明的一种基于核酸连接的新技术-LMO扩增。LMO技术是基于在短寡核苷酸不与模板杂交的条件下将一组短寡核苷酸连续连接到模板结合的引物上。我们展示了成功的扩增，足够的灵敏度，并对几种不同的扩增设计进行了评估。原型化验与现有的许多检测突变的技术一样快。第一阶段的结果为在第二阶段开发和标准化因子V研究试剂盒奠定了基础。我们将扩大这项工作，开发一种快速检测和在两阶段测试中区分三种可能的基因型别的试剂盒。这项测试将使用双荧光能量转移(FRET)分析。检测将基于从供体荧光团标记的低聚体到受体荧光团标记的低聚体的能量转移，这些低聚体通过连接-放大过程被结合。成功实施基于FRET的检测将大大缩短得出结果的时间。我们将利用在第一阶段获得的专业知识，开发另外两种与凝血障碍相关的常用突变检测方法，即凝血因子II和MTHFR。作为最终目标，我们将设计一种多路测试形式，同时为每一项测试筛选血液样本。使用不同颜色的荧光灯可以区分这三种突变。

DESCRIPTION (provided by applicant): Phase I of this proposal proved the feasibility of detection of the factor V Leiden mutation in human blood and correct determination of sample genotype as compared to a reference PCR method. The protocol used a new nucleic acid ligation-based technology, LMO amplification, invented at Lumigen. The LMO technology is based on the contiguous ligation of a set of short oligonucleotides to a template-bound primer under conditions in which the short oligonucleotides are not hybridized to the template. We demonstrated successful amplification, adequate sensitivity, and evaluated several different amplification designs. The prototype assay is as rapid as many existing techniques for detecting mutations. The Phase I results have laid the groundwork for the development and standardization of research kits for factor V using a microtiter plate format in Phase II. We will expand this work to develop a rapid test and kits for discrimination the three possible genotypes in a two-stage test. This test will utilize dual fluorescer energy transfer (FRET) assays. Detection will be based on energy transfer from a donor fluorophore-labeled oligomer to an acceptor fluorophore-labeled oligomer which become incorporated via the ligation-amplification process. Successful implementation of FRET-based detection will significantly shorten time to result. We will use the expertise gained in Phase I to develop two additional commonly used mutation tests related to clotting disorders, Factor II and MTHFR. As a final goal we will devise a multiplex test format for screening blood samples for each of these tests simultaneously. Use of different color fluorescers will distinguish the three mutations.