Genotyping and Haplotyping Using Thin Flim Biosensor Chips

使用薄生物传感器芯片进行基因分型和单体型分析

• 批准号: 6879910
• 负责人: DAVID C WARD
• 金额: $ 29.04万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879910
• 关键词: biosensor device; biotechnology; cell line; genetic screening; human genetic material tag; human population genetics; microarray technology; racial /ethnic difference; single nucleotide polymorphism; technology /technique development

The overall objective of this proposal is to better understand the genetic diversity among humans by defining the haplotype and linkage disequilibrium parameters at 1,200 single nucleotide polymorphism (SNP) sites in over 2,000 individuals representing 38 different racial/ethnic groups from around the world. This unique sample population, collected by Drs. K.K. and J.R. Kidd, provides the opportunity to not only characterize combinations of genetic variants relevant to disease-gene discovery but to describe the creation, maintenance and distribution of genotypes in the context of population genetic models. Approximately 900 hundred of the SNP sites map uniquely to a 12 Mb region in band 17q21 on human chromosome 17, while the remaining 300 map to twelve 200kb regions dispersed throughout the genome. A total of more than 2 x 10[6] SNP typings will be done with a novel assay platform developed by Dr. Zhong in the PIs laboratory. Allele-discriminating oligonucleotides first are arrayed and covalently attached to a 6x6 mm[2] silicon chip coated with a thin-film optical biosensor. Hybridization of target sequences (e.g. PCR amplicons) is then done in the presence of a mixture of biotinylated, SNP-specific oligomers and a thermostable DNA ligase. Selective ligation of biotinylated probe to the sequence matched (but not mismatched) capture probe is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin -horseradish peroxidase (HRP) conjugate and a precipitable HRP substrate. This assay is extremely robust, exhibits high sensitivity and specificity, is flexible (signals detected at low SNIP density without instrumentation or at high SNP density with an automated optical reader) and most importantly very economical. Additional studies will be done to improve the detection sensitivity of the biosensor SNP assay so that global SNP typing can be done with fragmented genomic DNA, thereby markedly increasing the number of SNPs that can be analyzed simultaneously and dramatically reducing the cost/SNP. Finally, we will use multiple displacement amplification (MDA), a novel whole genome DNA amplification procedure, in combination with rnicrodissection of a single metaphase chromosome (or chromosomal segments) to prepare DNA suitable for direct molecular haplotyping on biosensor chips.

这项建议的总体目标是通过定义1,200个单核苷酸多态(SNP)位点的单倍型和连锁不平衡参数来更好地了解人类的遗传多样性，这些SNP位点代表来自世界各地的38个不同种族/民族的2000多个个体。K.K.博士和J.R.Kidd博士收集的这一独特的样本群体，不仅提供了表征与疾病基因发现相关的遗传变异组合的机会，而且提供了在群体遗传模型的背景下描述基因类型的创建、维持和分布的机会。大约900个SNP位点唯一地定位到人类17号染色体17q21带上的12Mb区域，而其余300个SNP位点定位到分布在整个基因组中的12个200kb区域。使用钟博士在PIS实验室开发的新的分析平台，将进行超过2x10[6]个SNP分型。首先将识别等位基因的寡核苷酸排列并共价连接到涂有薄膜光学生物传感器的6x6 mm[2]硅芯片上。然后，在生物素化的、SNP特异的寡聚体和耐热的DNA连接酶的混合物中进行靶序列(例如，PCR扩增)的杂交。在与抗生物素-辣根过氧化物酶(HRP)结合物和可沉淀的HRP底物短暂孵育后，生物素化探针与序列匹配(但不是错配)的捕获探针的选择性连接显示为芯片表面的颜色变化(金色到蓝色/紫色)。这种检测方法非常可靠，表现出高灵敏度和高特异性，灵活(无需仪器在低片段密度下检测信号，或使用自动光学读取器在高SNP密度下检测信号)，最重要的是非常经济。将进行更多研究，以提高对 生物传感器SNP分析使全球SNP分型可以用片段基因组DNA进行，从而显著增加了可以同时分析的SNP数量，并显著降低了SNP的成本。最后，我们将使用多重置换扩增(MDA)这一新的全基因组DNA扩增方法，结合对单个中期染色体(或染色体片段)的显微切割，制备适合于在生物传感器芯片上直接进行分子单倍型分析的DNA。