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.

该提案的总体目的是通过在1,200多个代表38个不同种族/族裔群体的个人的个人中，在1,200个单核苷酸多态性（SNP）位点定义单倍型和连锁不平衡参数，以更好地了解人类之间的遗传多样性。这个独特的样本人群，由Drs收集。 K.K. J.R.基德（J.R. Kidd）提供了一个机会，不仅可以表征与疾病 - 基因发现相关的遗传变异的组合，还可以描述在人群遗传模型的背景下的基因型的创造，维持和分布。大约90000个SNP站点在人类17中的17q21中独特地映射到12 MB区域，而其余的300个地图则分散在整个基因组中。总共超过2 x 10 [6] SNP键入将通过Zhong博士在PIS实验室开发的新型测定平台进行。首先将等位基因 - 歧义寡核苷酸固定并共价连接到6x6 mm [2]硅芯片上，该硅芯片涂有薄膜光学生物传感器。然后在存在生物素化的，SNP特异性的低聚物和可热稳定DNA连接酶的混合物的情况下进行靶序列（例如PCR扩增子）的杂交。在与抗生物素 - 棘手过氧化物酶（HRP）结合物和可降低的HRP substrate的HRP substrate中短暂孵育后，将生物素化探针与匹配（但不匹配）捕获探针匹配（但不匹配）捕获探针的选择性结扎。该测定非常强大，具有高灵敏度和特异性，是柔性的（在没有仪器的情况下以低剪切密度检测到信号，或者在具有自动化光学读取器的高SNP密度下），最重要的是非常经济。将进行其他研究以提高检测灵敏度 生物传感器SNP测定法可以使用碎片的基因组DNA进行全局SNP键入，从而显着增加了可以同时和大幅度分析的SNP数量，从而大大降低了成本/SNP。最后，我们将使用多个位移扩增（MDA），这是一种新型的整个基因组DNA扩增程序，结合单个中期染色体（或染色体段）的r nicroRycouse，准备适合于生物传感器芯片上直接分子单倍分型的DNA。