Band M, Ron M
Institute of Animal Sciences, Volcani Center, Bet Dagan, Israel.
Anim Biotechnol. 1998;9(1):35-45. doi: 10.1080/10495399809525890.
Three sets of fluorescent labelled primers were used to amplify bovine trinucleotide microsatellites from DNA pools. DNA from 20 individuals were collected to create 3 pools differing in allele frequencies. Replicate mixes from each pool were used as template for PCR reactions. PCR products were separated and quantified on an automated DNA sequencer. Allele frequency estimates from pooled samples corrected for overlapping shadow peaks were calculated. Rare alleles representing only 2.5% of the total pool were accurately detected. Standard error of allele frequency estimates expressed as percent of the total 40 chromosomes per pool ranged between 0.8%-4.6% for different microsatellite-pool combinations as compared to 8.0% binomial sampling error. Regression coefficients of actual allele frequencies, determined by individual genotyping, on estimated frequencies ranged from 0.96-1.06. As regression slopes were close to unity it can be deduced that corrected peak height values from a DNA pool are unbiased estimates of actual allele frequencies. With standard error of the y-intercept of 0.21, the 95% confidence interval of allele frequency is 0.42 alleles or 1% in a pool of 40 chromosomes. Thus, it would be possible to detect an allele with a frequency of greater than 1% within the pool.
使用三组荧光标记引物从DNA池中扩增牛三核苷酸微卫星。收集来自20个个体的DNA以创建3个等位基因频率不同的池。每个池的重复混合物用作PCR反应的模板。PCR产物在自动DNA测序仪上进行分离和定量。计算校正了重叠阴影峰的合并样本的等位基因频率估计值。准确检测到仅占总池2.5%的稀有等位基因。与8.0%的二项式抽样误差相比，不同微卫星-池组合的等位基因频率估计标准误差以每个池40条染色体总数的百分比表示，范围在0.8%-4.6%之间。通过个体基因分型确定的实际等位基因频率与估计频率的回归系数范围为0.96-1.06。由于回归斜率接近1，可以推断DNA池校正后的峰高值是实际等位基因频率的无偏估计。y轴截距的标准误差为0.21，等位基因频率的95%置信区间为0.42个等位基因，即40条染色体的池中为1%。因此，有可能在池中检测到频率大于1%的等位基因。
