Genotyping and Haplotyping Using Thin Flim Biosensor Chips

本提案的总体目标是通过定义来自世界各地38个不同种族/民族的2000多个个体的1200个单核苷酸多态性（SNP）位点的单倍型和连锁不平衡参数，更好地了解人类之间的遗传多样性。这个独特的样本群体，由dr。K.K.和J.R.基德，提供了一个机会，不仅表征与疾病基因发现相关的遗传变异组合，而且描述了在群体遗传模型背景下基因型的创造、维持和分布。大约900个SNP位点唯一地映射到人类17号染色体17q21带的12 Mb区域，而其余300个位点映射到分散在整个基因组中的12个200kb区域。钟博士在PIs实验室开发的新型检测平台将完成总计超过2 × 10[6]个SNP分型。等位基因鉴别寡核苷酸首先被排列并以共价连接到涂有薄膜光学生物传感器的6x6mm[2]硅片上。然后在生物素化、snp特异性低聚物和耐热性DNA连接酶的混合物的存在下进行目标序列的杂交（例如PCR扩增子）。与抗生物素-辣根过氧化物酶（HRP）偶联物和可沉淀的HRP底物短暂孵育后，生物素化探针与序列匹配（但不是不匹配）的捕获探针选择性连接，可见芯片表面的颜色变化（金色到蓝色/紫色）。该分析非常稳健，具有高灵敏度和特异性，灵活（在低SNP密度下无需仪器检测信号或在高SNP密度下使用自动光学读取器检测信号），最重要的是非常经济。将进行进一步的研究以提高该方法的检测灵敏度

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.