High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR
High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR
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DOI：
10.1101/gr.10.2.258
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发表时间：
2000-02-01
期刊：
影响因子：
7
通讯作者：
Higuchi, R
中科院分区：
文献类型：
--
作者：
Germer, S;Holland, MJ;Higuchi, R
我们开发了一种准确、廉价且高通量的方法，用于确定 DNA 样本池中双等位基因多态性的等位基因频率。该检测将动态（实时定量）PCR 与等位基因特异性扩增相结合，无需 PCR 后处理。对样品中每个等位基因的相对量进行定量。这是通过在两个单独的 PCR 反应之间划分等份的汇集 DNA 来完成的，每个反应都包含一个对一种或另一种等位基因 SNP 变体具有特异性的引物桶。对于具有等量两个等位基因的池，两次扩增应在相同的循环数下达到可检测的荧光水平。对于包含不等比例的两个等位基因的库，需要使用两个扩增反应之间的循环数差异来计算相对等位基因量。我们在已知预定 SNP 等位基因频率为 5% 至 95% 的样本上证明了该测定的准确性和可靠性，其中包括总共使用八种不同 SNP 的人类和小鼠 DNA 库。测量已知等位基因频率的准确性非常高，测量频率和已知频率之间的相关性强度为 r(2)=0.997。对于多达 1000 个群体样本，与仅由抽样误差造成的灵敏度损失相比，由测量误差造成的灵敏度损失通常很小。我们相信，通过提供一种同时、廉价且可重复地对数千个样本进行 SNP 基因分型的方法，该方法是检测候选基因关联研究和全基因组连锁不平衡扫描中有意义的多态性差异的强大策略。
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 pail 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 call 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.