雙鏈置換探針實時PCR用於DNA池的等位基因頻率測定

結合熒光雙鏈置換探針的特異性和實時PCR的準確定量能力，建立了一種新穎、準確、價廉且高通量的測定DNA池等位基因頻率的方法。該方法採用不同熒光標記的雙鏈置換探針1次PCR反應即可測定出等位基因頻率。實驗以β地中海貧血CDs41 42（ TCTT）突變為對象，分別用熒光染料FAM和ROX標記野生型和突變型探針，由實時PCR檢測建立的等位基因濃度與循環閾值的響應曲線計算DNA池的等位基因頻率。結果表明，建立的系列等位元基因濃度與循環閾值呈線性關係，線性相關系數分別為0.9977（野生型等位基因）和0.9938（突變型等位基因），可檢測的最低等位基因頻率達1%，等位基因頻率在1%～90%範圍內測定誤差小於4%。該方法可廣泛用於流行病學調查、遺傳連鎖分析以及全基因組連鎖不平衡掃描等領域。
An accurate, inex pensive 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 show ed 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 low est 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 pow erful strategy for large population-based epidemiology study, detecting meaningful polymorphic differences in candidate gene association studies, and genome-wide linkage disequilibrium scans. Copyright © 2004 廈門大學.