Whole genome amplification and DNA pooling strategies

全基因组扩增和 DNA 混合策略

• 批准号: 6650468
• 负责人: Immaculata De Vivo
• 金额: $ 8.17万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6650468
• 关键词: DNA; epidemiology; gene frequency; genetic polymorphism; genetic screening; genome; genotype; human data; human tissue; nucleic acid amplification techniques; questionnaires; single nucleotide polymorphism

DESCRIPTION (provided by applicant): We propose to assess the utility of a recently developed variant of the whole genome amplification technique, multiple displacement amplification (MDA), for high-throughput genotyping of DNA samples for molecular epidemiology studies. We hypothesize that MDA products will be ideal for genotyping analysis when consistent DNA concentrations are required from samples that may be of uncertain concentrations and quality. To this end, we will amplify and genotype by various techniques a subset of buffy coat and buccal cell DNA samples ranging from low to high DNA concentrations from the Nurses Health Study (NHS) and the Health Professional Follow-up Study (HPFS) cohorts. Our genotyping analysis will include microsatellites, insertion/deletions and single nucleotide polymorphisms (SNPs) in GC-rich regions, markers that can be challenging to genotype. In addition, we will assess the utility of DNA pooling strategies to estimate allele frequencies in large nested case-control studies. We will use both DNA sources from the NHS and HPFS, as well as MDA-generated products to determine whether allele frequencies in pooled DNA will correlate strongly with those measured in individuals across a wide range of allele frequencies. Results from the pooling experiments will determine whether this method may be sensitive enough for use in nested case-control studies where a small difference in allele frequency is expected.

描述（由申请人提供）： 我们建议评估最近开发的全基因组扩增技术，多重置换扩增（MDA）的变种，用于分子流行病学研究的DNA样本的高通量基因分型的效用。我们假设，MDA产品将是理想的基因分型分析时，一致的DNA浓度需要从样本，可能是不确定的浓度和质量。为此，我们将通过各种技术对来自护士健康研究（NHS）和健康专业随访研究（HPFS）队列的血沉棕黄层和颊细胞DNA样本的子集进行扩增和基因分型，DNA浓度从低到高。我们的基因分型分析将包括微卫星，插入/缺失和单核苷酸多态性（SNP）在GC丰富的地区，标志物，可以是具有挑战性的基因分型。此外，我们还将评估DNA合并策略在大型巢式病例对照研究中估计等位基因频率的实用性。我们将使用来自NHS和HPFS的DNA来源以及MDA生成的产品来确定合并DNA中的等位基因频率是否与在广泛的等位基因频率范围内的个体中测量的等位基因频率密切相关。合并实验的结果将确定该方法是否足够灵敏，可用于预期等位基因频率差异较小的巢式病例对照研究。