SNP Detection with Unlabeled, Unamplified Target DNA

描述(申请人提供)：该研究项目的目的是将双重杂交诊断基因分型方法进一步发展为能够使用未标记、未扩增的DNA作为目标的多重分析方法。在第一阶段，利用扩增的靶标和单一的荧光团标记证明了双重杂交方法的可行性。所有结果表明，该方法很容易转变为一种利用剪切基因组DNA和荧光微球标记的并行、高灵敏度的方法。目前的基因诊断测试费用很高，通常只在出现不良症状后对少数特定突变进行检测。这种诊断测试的简单性将消除目前获取大量遗传信息所存在的成本限制，使常规诊断成为预防性药物。具体的改进包括：1)操作时间更短，操作难度更小(不需要聚合酶链式反应、样本标记或多重等位分析)，2)减少患者样本(血液、组织等)，以及3)SNPs和更大突变的并行基因分型。该方法使用目标分子的双重杂交。一个杂交事件发生在基因组DNA样本中的目标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，一个基因的所有等位基因都将定位在一个阵列位置。另一个杂交事件发生在目标核酸和肽核酸(PNA，一种核酸类似物)的短探针之间，后者与DNA形成稳定的、高度序列特异性的杂交。每个等位基因变异都有自己的PNA探针，带有唯一的标记。在探针位置检测到的标记类型表明患者基因组中存在的等位基因。这一过程非常灵活，因为几乎可以使用任何标签。混合标记的PNA探针、DNA探针阵列，以及可能的缓冲器和自动流动池(能够保持正确的温度和液体处理)将出售给消费者。对于第二阶段，将设计一种对怀疑影响结肠癌易感性的SNPs进行基因分型的阵列。在FDA批准该方法用于诊断之前，该研究工具和其他定制阵列可以用于研究用途。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的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.