A Surface Invader Assay for SNP Analysis on DNA Arrays

描述（申请人摘要）：本提案的目标是开发一种 单核苷酸多态性平行评分的单步方法 （SNPs）以表面阵列格式进行。该方法基于最近 开发了用于SNP评分的侵入性切割测定，称为“侵入者”， （Third Wave Technologies，Inc.）因为它是一种信号放大 技术而不是靶扩增技术，该测定法不是 受到PCR特有的污染/遗留问题的影响。这是一个 非常简单、稳健和等温分析，非常适合高通量 分析.在所提出的基于表面的程序版本（“表面 侵入测定”），添加到含有靶人类基因组DNA的表面， 给定的SNP等位基因将导致相应的SNP的特异性切割， 含有荧光团猝灭剂染料表面固定的探针寡核苷酸 汇率这种切割将发生在荧光团和猝灭剂之间， 留下未猝灭的荧光团附着在表面上。荧光 该未猝灭荧光团的强度将基本上大于 猝灭的荧光团的区域，从而DNA阵列的区域 含有该探针寡核苷酸将显示出增加的荧光 以特定的靶依赖性方式的强度。通过使用这样的数组， 探针寡核苷酸，每个待分型的SNP等位基因一个，单次添加 沿着其他所需的测定， 试剂（缓冲液和酶），然后孵育、洗涤和荧光 成像步骤将产生针对每个SNP分析的目标DNA的基因型 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.