Nazarenko I A, Bhatnagar S K, Hohman R J
Oncor, Inc., 209 Perry Parkway, Gaithersburg, MD 20877, USA.
Nucleic Acids Res. 1997 Jun 15;25(12):2516-21. doi: 10.1093/nar/25.12.2516.
A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.
本文描述了一种在封闭系统中直接检测PCR扩增DNA的新方法。该方法基于将能量转移标记的引物掺入扩增产物中。PCR引物在其5'端含有发夹结构，供体和受体部分位于发夹茎上彼此靠近的位置。引物的设计方式使得仅当引物掺入扩增产物时才产生荧光信号。使用以荧光素作为供体和4-（4'-二甲基氨基苯基偶氮）苯甲酸（DABCYL）作为猝灭剂标记的发夹引物，获得了35:1的信号背景比。修饰的发夹引物不干扰DNA聚合酶的活性，并且可以使用耐热的Pfu和Taq聚合酶。该方法应用于前列腺特异性抗原cDNA的检测。结果表明，扩增产物的荧光强度与掺入引物的量相关，并且最初模板少至10个分子即可被检测到。该技术消除了污染残留的风险，简化了扩增检测，并为在极宽的动态范围内对扩增DNA进行实时定量开辟了新的可能性。
