Molecular Diagnostic Assays for Genetic Diseases

描述（由申请方提供）：与参考PCR方法相比，该提案的I期证明了检测人血液中凝血因子V Leiden突变和正确测定样本基因型的可行性。该方案使用了一种新的基于核酸连接的技术，LMO扩增，由Lumigen发明。LMO技术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至模板结合的引物。我们证明了成功的扩增，足够的灵敏度，并评估了几种不同的扩增设计。原型检测与许多现有的检测突变的技术一样快速。第一阶段的结果奠定了基础的发展和标准化的研究试剂盒的第二阶段使用微量滴定板格式的因子V。我们将扩大这项工作，以开发一种快速测试和试剂盒，用于在两阶段测试中区分三种可能的基因型。本试验将使用双荧光能量转移（FRET）试验。检测将基于从供体荧光团标记的寡聚体到受体荧光团标记的寡聚体的能量转移，所述受体荧光团标记的寡聚体通过连接-扩增过程并入。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I期获得的专业知识，开发另外两种与凝血障碍相关的常用突变试验，即因子II和MTHFR。作为最终目标，我们将设计一种多重检测形式，用于同时筛查这些检测中的每一种血液样本。使用不同颜色的荧光剂将区分三种突变。

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.