A Surface Invader Assay for SNP Analysis on DNA Arrays

描述(申请人摘要)：这项提案的目标是开发一种 单核苷酸多态并行评分的单步法 (SNPs)表面阵列格式。这种方法是基于最近的一项 开发了用于SNP评分的侵入性切割试验，称为“入侵者” 测试(Third Wave Technologies，Inc.)因为它是一种信号放大 技术而不是靶扩增技术，这种检测不是 受制于聚合酶链式反应特有的污染/携带问题。这是一个 非常简单、坚固和恒温的分析非常适合高通量 分析。在拟议的基于表面的程序版本(“表面”)中 入侵者检测“)，添加到目标人类基因组DNA表面，含有 给定的SNP等位基因会导致相应的 含荧光猝灭染料的表面固定化探针寡核苷酸 一对。这种切割将发生在荧光团和猝灭剂之间， 使未淬灭的荧光团附着在表面上。荧光 这种未淬灭的荧光团的强度将大大超过 猝灭的荧光团，从而DNA阵列的区域 含有该探针的寡核苷酸将显示出增强的荧光 强度以特定的依赖于目标的方式。通过使用一组这样的 探针寡核苷酸，每个SNP等位基因对应一个待分型，一次加成 将目标人类基因组DNA连同其他所需的分析一起送到表面 试剂(缓冲液和酶)，然后孵育、洗涤和荧光 成像步骤，将产生为每个SNP分析的目标DNA的基因类型 在数组中表示。

DESCRIPTION (Applicant's Abstract): The goal of this proposal is to develop a single step method for the parallel scoring of single nucleotide polymorphisms (SNPs) in a surface array format. The approach is based upon a recently developed invasive cleavage assay for SNP scoring referred to as the 'Invader" assay (Third Wave Technologies, Inc.) As it is a signal amplification technology rather than a target amplification technology, this assay is not subject to the contamination/carryover problems characteristic of PCR. It is a very simple, robust, and isothermal assay well-suited for high throughput analysis. In the proposed surface-based version of the procedure (the "Surface Invader Assay"), addition to the surface of target human genomic DNA containing a given SNP allele will result in specific cleavage of a corresponding surface-immobilized probe oligonucleotide containing a fluorophore-quencher dye pair. This cleavage will occur between the fluorophore and the quencher, leaving the unquenched fluorophore attached to the surface. The fluorescence intensity of this unquenched fluorophore will be substantially greater than that of the quenched fluorophore, and thus the region of the DNA array containing that probe oligonucleotide will exhibit increased fluorescence intensity in a specific target-dependent fashion. By employing an array of such probe oligonucleotides, one for each SNP allele to be typed, a single addition of target human genomic DNA to the surface, along with the other needed assay reagents (buffer and enzyme), followed by incubation, washing, and fluorescence imaging steps, will yield the genotypes of the target DNA analyzed for each SNP represented in the array.