摘要
结合荧光双链置换探针的特异性和实时PCR的准确定量能力,建立了一种新颖、准确、价廉且高通量的测定DNA池等位基因频率的方法.该方法采用不同荧光标记的双链置换探针1次PCR反应即可测定出等位基因频率.实验以β 地中海贫血CDs41 42( TCTT)突变为对象,分别用荧光染料FAM和ROX标记野生型和突变型探针,由实时PCR检测建立的等位基因浓度与循环阈值的响应曲线计算DNA池的等位基因频率.结果表明,建立的系列等位基因浓度与循环阈值呈线性关系,线性相关系数分别为0.9977(野生型等位基因)和0.9938(突变型等位基因),可检测的最低等位基因频率达1%,等位基因频率在1%~90%范围内测定误差小于4%.该方法可广泛用于流行病学调查、遗传连锁分析以及全基因组连锁不平衡扫描等领域.
An accurate,inexpensive and high-throughput method for determining the allele frequency of biallelic polymophisms in pools of DNA samples was developed. The assay combined the high specificity of double-stranded displacing probes with real-time quantitative PCR.Using differently labeled probes, the relative amounts of each allele in a sample could be quantified in one reaction. This method was evaluated using CDs41-42(-TCTT) mutation in human β-globin gene causative for β-thalassemia as an example. The wild-type and mutant probes were labeled with FAM and ROX,respectively,and were recruited in one reaction. Using the constructed DNA pools, real time PCR was performed to establish the relationship between the relative amount of each allele and the threshold cycle value.With this relationship, allele frequency of the pooled DNA sample could be obtained by its threshold cycle value and the DNA concentration of the sample. The results showed that there is a linear relationship between the amount of the allele and the threshold cycle value through the whole allele frequency range studied,and the lowest allele frequency detected was 1%.The linear correlation coefficient was 0.997 7 for wile-type allele and 0.993 8 for mutant allele,respectively. Within the allele frequency range of 1%~90%,the relative error calculated was less than 4%,which was acceptable for allele frequency determination in pooled DNA samples.Considering its simplicity,reliability,and low cost,this approach was a powerful strategy for large population-based epidemiology study,detecting meaningful polymorphic differences in candidate gene association studies, and genome-wide linkage disequilibrium scans.
出处
《厦门大学学报(自然科学版)》
CAS
CSCD
北大核心
2004年第B08期113-118,共6页
Journal of Xiamen University:Natural Science
基金
国家自然科学基金资助项目(30170834)
福建省自然科学基金重点项目(C0220001)资助.