Germer S, Holland M J, Higuchi R
Roche Molecular Systems, Alameda, California 94501 USA.
Genome Res. 2000 Feb;10(2):258-66. doi: 10.1101/gr.10.2.258.
We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r(2) = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.
我们开发了一种准确、廉价且高通量的方法，用于测定DNA样本池中二等位基因多态性的等位基因频率。该检测方法将动力学（实时定量）PCR与等位基因特异性扩增相结合，无需PCR后处理。对样本中每个等位基因的相对量进行定量。这通过将等份的混合DNA在两个单独的PCR反应之间分配来进行，每个反应都包含一对针对一个或另一个等位基因SNP变体的引物。对于两个等位基因含量相等的样本池，两个扩增反应应在相同的循环数达到可检测的荧光水平。对于两个等位基因比例不相等的样本池，两个扩增反应之间的循环数差异可用于计算相对等位基因量。我们使用总共八个不同的SNP，在已知预定SNP等位基因频率为5%至95%的样本上，包括人和小鼠DNA样本池，证明了该检测方法的准确性和可靠性。测量已知等位基因频率的准确性非常高，测量频率与已知频率之间的相关强度r(2)=0.997。与仅由抽样误差导致的灵敏度损失相比，对于多达1000个的群体样本，测量误差导致的灵敏度损失通常最小。我们相信，通过提供一种同时对多达数千个样本进行SNP基因分型的方法，该方法廉价且可重复，是在候选基因关联研究和全基因组连锁不平衡扫描中检测有意义的多态性差异的有力策略。
