Whole genome amplification and DNA pooling strategies

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的一种变体，多重置换扩增(MDA)，用于高通量DNA样本的基因分型，用于分子流行病学研究。我们假设，当需要从可能具有不确定浓度和质量的样本中获得一致的DNA浓度时，丙二醛产品将是进行基因分型分析的理想产品。为此，我们将通过各种技术从护士健康研究(NHS)和健康专业人员后续研究(HPFS)队列中扩增和分型一组从低DNA浓度到高DNA浓度的巴菲毛皮和口腔细胞DNA样本。我们的基因分型分析将包括GC富集区的微卫星、插入/缺失和单核苷酸多态(SNPs)，这些标记可能对基因分型构成挑战。此外，我们将评估DNA池策略在大型嵌套病例对照研究中估计等位基因频率的有效性。我们将使用NHS和HPFS的DNA来源以及丙二醛生成的产品来确定池DNA中的等位基因频率是否会与在广泛等位基因频率范围内在个体中测量的等位基因频率强烈相关。合并实验的结果将决定该方法是否足够敏感，以用于预期等位基因频率存在微小差异的嵌套病例对照研究。

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.