SNP Detection with Unlabeled, Unamplified Target DNA

使用未标记、未扩增的目标 DNA 进行 SNP 检测

• 批准号: 6643759
• 负责人: RICHARD Ashley HURT
• 金额: $ 37.67万
• 依托单位: ATOM SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6643759
• 关键词: DNA; colon neoplasms; genotype; method development; microarray technology; nucleic acid hybridization; nucleic acid probes; nucleic acid quantitation /detection; peptide nucleic acids; single nucleotide polymorphism; technology /technique development

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.

描述（由申请人提供）：该研究项目的目的是进一步发展双杂交诊断基因分型方法，使其成为能够使用未标记，未扩增的DNA作为目标的多重分析方法。双杂交方法的可行性在第一阶段通过扩增的靶标和单荧光团标记进行了验证。所有结果表明，该方法可以很容易地转换为使用剪切基因组DNA和荧光微球标记的平行，高灵敏度的方法。目前的基因诊断检测价格昂贵，而且通常只对出现不良症状后的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在获取大量遗传信息方面存在的成本限制，使常规诊断成为预防性医学。特别的改进包括：1)操作时间缩短，操作难度降低（不需要PCR、样品标记或多次aliquote）， 2)减少患者采样（血液、组织等），3)snp和较大突变的平行基因分型。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。这种杂交的结果是目标核酸的序列特异性固定。例如，一个基因的所有等位基因将定位到一个阵列位点。另一个杂交事件发生在靶核酸和短探针的肽核酸（PNA，一种核酸类似物）之间，与DNA形成稳定的、高度序列特异性的杂交。每个等位基因变体都有自己的带有唯一标签的PNA探针。在探针位点检测到的标记类型表明存在于患者基因组中的等位基因。这个过程是高度灵活的，因为几乎任何标签都可以使用。汇集标记的PNA探针，DNA探针阵列，可能还有缓冲液和自动流动池（能够保持正确的温度和液体处理）将出售给消费者。在第二阶段，将设计一个基因分型阵列，用于怀疑影响结肠癌易感性的snp。该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，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。