用荧光双链引物特异扩增并定量核酸

Specific and Quantitative Assay for DNA Sequence Amplification by Using a Doubled Stranded Primer

描述了一种利用特殊的双链引物———突触引物 ,用于核酸扩增的特异定量检测 .在传统的引物的 5’端标记荧光物质 ,而在引物的互补序列的 3’端标记荧光淬灭剂以封闭其延伸 .二者杂交即成双链突触引物 .在制备PCR反应混合物阶段以及加热的初期 ,突触引物保持稳定的双链结构而不能引导扩增 ,在退火阶段 ,突触引物部分解链导致引物延伸 ,荧光物质与淬灭剂分开而产生荧光 .利用人 β珠蛋白基因对此方法进行了验证 .这种可自行退火的荧光引物不仅简单地实现了实时扩增 。

A simple, specific and quantitative assay for DNA sequence amplification by using a doubled stranded primer called antenna primer is described. To construct antenna primer, the conventional primer was labeled with a fluorophor at its 5'-end, and a complementary sequence of the primer was labeled at 3'-end with a dark fluorescent quencher to block its extension, and non-fluorescent double-strand primer was formed upon hybridization. During preparation of the reaction mixture and initial heating, the antenna primer has a stable duplex structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature, it partially melted and primer extension began and fluorescence was produced. The method has been validated with amplification of human β-globulin gene. The antenna primer is a novel design. The self-annealing fluorogenic primers not only realize real-time amplification in a simple way, but also offer improved specificity and more robust synthesis than hot start PCR.