Wolford J K, Blunt D, Ballecer C, Prochazka M
Clinical Diabetes and Nutrition Section, Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institues of Health, Phoenix, AZ 85016, USA.
Hum Genet. 2000 Nov;107(5):483-7. doi: 10.1007/s004390000396.
One of the critical steps in the positional cloning of a complex disease gene involves association analysis between a phenotype and a set of densely spaced diallelic markers, typically single nucleotide repeats (SNPs), covering the region of interest. However, the effort and cost of detecting sufficient numbers of SNPs across relatively large physical distances represents a significant rate-limiting step. We have explored DNA pooling, in conjunction with denaturing high performance liquid chromatography (DHPLC), as a possible strategy for augmenting the efficiency, economy, and throughput of SNP detection. DHPLC is traditionally used to detect variants in polymerase chain reaction products containing both allelic forms of a polymorphism (e.g., heterozygotes or a 1:1 mix of both alleles) via heteroduplex separation and thereby requires separate analyses of multiple individual test samples. We have adapted this technology to identify variants in pooled DNA. To evaluate the utility and sensitivity of this approach, we constructed DNA pools comprised of 20 previously genotyped individuals with a frequency representation of 0%-50% for the variant allele. Mutation detection was performed by using temperature-modulated heteroduplex formation/DHPLC and dye-terminator sequencing. Using DHPLC, we could consistently detect SNPs at lower than 5% frequency, corresponding to the detection of one variant allele in a pool of 20 alleles. In contrast, fluorescent sequencing detected variants in the same pools only if the frequency of the less common allele was at least 10%. We conclude that DNA pooling of samples for DHPLC analysis is an effective way to increase throughput efficiency of SNP detection.
复杂疾病基因定位克隆的关键步骤之一，是在一种表型与一组紧密排列的双等位基因标记（通常为单核苷酸重复序列，即SNP）之间进行关联分析，这些标记覆盖了感兴趣的区域。然而，在相对较大的物理距离上检测足够数量SNP的工作量和成本，是一个显著的限速步骤。我们探索了将DNA池与变性高效液相色谱（DHPLC）相结合，作为提高SNP检测效率、经济性和通量的一种可能策略。传统上，DHPLC用于通过异源双链分离来检测聚合酶链反应产物中的变异体，这些产物包含多态性的两种等位基因形式（例如杂合子或两种等位基因的1:1混合），因此需要对多个单独的测试样品进行单独分析。我们对这项技术进行了改进，以识别混合DNA中的变异体。为了评估这种方法的实用性和敏感性，我们构建了由20个先前已进行基因分型的个体组成的DNA池，变异等位基因的频率代表为0%-50%。通过使用温度调制异源双链形成/DHPLC和染料终止测序进行突变检测。使用DHPLC，我们能够始终如一地检测频率低于5%的SNP，这相当于在20个等位基因的池中检测到一个变异等位基因。相比之下，荧光测序只有在较不常见等位基因的频率至少为10%时，才能检测到同一池中的变异体。我们得出结论，用于DHPLC分析的样品DNA池是提高SNP检测通量效率的有效方法。
