SNP Detection with Unlabeled, Unamplified Target DNA

描述（由申请人提供）：本研究项目的目的是将双重杂交诊断基因分型方法进一步开发为能够使用未标记、未扩增DNA作为靶标的多重检测方法。双杂交方法的可行性在第一阶段使用扩增的目标和单荧光标记进行了证明。所有结果表明，该方法将容易地转移到一个平行的，高灵敏度的方法，使用剪切的基因组DNA和荧光微球标记。目前的基因诊断测试是昂贵的，通常只进行了一些特定的突变后，出现不良症状。这种诊断测试的简单性将消除成本限制，目前存在的获得大量遗传信息，使常规诊断成为预防性药物。具体改进包括：1）更短的操作时间和更少的执行程序的困难（不需要PCR、样品标记或多次等分），2）减少患者取样（血液、组织等），和3）SNP和较大突变的平行基因分型。该方法使用靶分子的双重杂交。一个杂交事件发生在基因组DNA样品中的靶片段和固定在阵列中的长DNA探针之间。这种杂交的结果是靶核酸的序列特异性固定。例如，基因的所有等位基因将定位于一个阵列位点。另一个杂交事件发生在靶核酸和肽核酸（PNA，核酸类似物）的短探针之间，肽核酸与DNA形成稳定的、高度序列特异性的杂交体。每个等位基因变体具有其自身的具有独特标记的PNA探针。在探针位点检测到的标记类型指示患者基因组中存在的等位基因。该过程非常灵活，因为几乎可以使用任何标签。将向消费者出售合并的标记PNA探针、DNA探针阵列以及可能的缓冲液和自动流动池（能够保持正确的温度和液体处理）。对于II期，将设计用于对怀疑影响结肠癌易感性的SNP进行基因分型的阵列。这种研究工具和其他定制阵列可以出售用于研究用途，直到该方法可以被FDA批准用于诊断用途。该设备的一个主要研究市场将是确定疾病的SNP模式，其中必须对每种疾病的数千名患者进行大规模平行SNP基因分型。

DESCRIPTION (provided by applicant): The aim of the research project is to further develop the dual hybridization diagnostic genotyping method into a multiplex assay capable of using unlabeled, unamplified DNA as the target. The feasibility of the dual hybridization method was demonstrated in Phase I using amplified targets and single fluorophore labels. All results indicate that the method will transfer easily to a parallel, high sensitivity method using sheared genomic DNA and fluorescent microsphere labels. Current genetic diagnostic tests are expensive and usually only performed for a few specific mutations after adverse symptoms have occurred. The simplicity of this diagnostic test will remove the cost limitation, which currently exists for obtaining large amounts of genetic information, allowing routine diagnostics to be preventative medicine. Particular improvements include: 1) shorter manipulation time and less difficulty performing the procedure (no PCR, sample labeling, or multiple aliquoting is required), 2) reduction of patient sampling (blood, tissue, etc.), and 3) parallel genotyping of SNPs and larger mutations. The method uses a dual hybridization of target molecules. One hybridization event occurs between targeted fragments in the genomic DNA sample and long DNA probes immobilized in an array. The result of this hybridization is a sequence-specific immobilization of target nucleic acids. For example, all alleles of a gene would be localized to one array site. The other hybridization event occurs between the target nucleic acid and short probes of peptide nucleic acid (PNA, a nucleic acid analog) that form stable, highly sequence-specific hybrids with DNA. Each allelic variant has its own PNA probe with a unique label. The types of labels detected at the probe site indicate the alleles present in the patient's genome. This procedure is highly flexible since almost any label can be used. Pooled labeled PNA probes, DNA probe arrays, and possibly buffers and an automated flow cell (capable of maintaining correct temperature and liquid handling) will be sold to the consumer. For Phase II, an array for genotyping SNPs suspected of influencing susceptibility to colon cancer will be designed. This research tool and other custom arrays can be sold for research use until the method can be approved by the FDA for diagnostic use. A major research market for the device would be determining SNP patterns for diseases where massively parallel SNP genotyping must be performed on thousands of patients for each disease.