HYBRIDIZATION PROXIMITY ASSAY (HYPA)

基因突变可以引起等位基因的变异，导致遗传性疾病，并使受影响的人容易患病。基因突变的常规检测是一项既耗时又繁琐的任务，因为大多数方法都需要使用高分辨率凝胶电泳法。相比之下，基于发光的分析更快速，更容易实现高通量操作的自动化。在即将开发的分析中，通过适当的发光探针之间的共振能量转移，可以促进在跨越数百个碱基对的区域中检测单个核苷酸的突变。通过使用最近开发的探针，避免了与以前的荧光分析有关的问题。传统染料的短寿命荧光必须与生物样品的光散射和自身荧光的高背景竞争，而基于镧系元素的探针的时间分辨荧光消除了这些背景问题。通过使用发夹探针进一步降低本底，该发夹探针被淬灭，直到杂交到正确的靶序列。因此，建议的检测方法可直接用于患者样本或聚合酶链式反应扩增产物。在这些微量分析中筛选大量样品的能力将有利于生物芯片开发、组织分型、基因表达、多重测序和遗传疾病测试等领域。这些加强的分析将对卫生保健成本效率、疾病的早期诊断、治疗和预防产生影响。拟议的商业应用：这项技术将提供探针，在与目标杂交时发出敏感的、低背景的时间分辨荧光信号，使其成为生物芯片应用的理想选择，用于同时进行序列微采样和突变检测。应用包括遗传病的鉴定、等位基因的组织分类和多重杂交测序。

Genetic mutations can give rise to variations in alleles, cause genetic diseases, and predispose affected individuals to illnesses. Routine detection of genetic mutations is a time-consuming and tedious task, since most methods require the use of high resolution gel electrophoresis. In contrast, luminescence-based assays are more rapid and easily automated for high throughput operation. In the assays to be developed, detecting mutations of a single nucleotide in regions spanning hundreds of base pairs may be facilitated by resonance energy transfer between appropriate luminescent probes. Problems associated with previous fluorescence assays are circumvented by the use of recently developed probes. While short-lived fluorescence from conventional dyes must compete with high backgrounds from light scattering and autofluorescence of biological specimens, time-resolved fluorescence of lanthanide-based probes eliminate these background problems. Background is reduced further by using hairpin probes that are quenched until hybridized to the correct target sequence. Therefore, the proposed assays may be used directly on samples from patients or PCR-amplified products. The ability to screen large numbers of samples in these microassays will benefit areas of biochip development, tissue typing, gene expression, multiplex sequencing, and genetic disease testing. These enhanced assays will have impacts on health care cost efficiency, early diagnosis, treatment, and prevention of diseases. PROPOSED COMMERCIAL APPLICATIONS: This technology will provide probes which emit sensitive, low-background time-resolved fluorescence signals upon hybridization to targets, making it ideal for biochip applications for simultaneous microsampling of sequence and detection of mutations. Applications include identification of genetic diseases, tissue typing of alleles, and multiplex hybridization sequencing.