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首页> 外文期刊>Nucleic Acids Research >A CLOSED TUBE FORMAT FOR AMPLIFICATION AND DETECTION OF DNA BASED ON ENERGY TRANSFER
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A CLOSED TUBE FORMAT FOR AMPLIFICATION AND DETECTION OF DNA BASED ON ENERGY TRANSFER

机译：基于能量转移的扩增和检测DNA的封闭管格式

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摘要

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.
机译：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，供体和受体部分紧密位于发夹茎上。以这样的方式设计引物：仅当将引物掺入扩增产物中时才产生荧光信号。使用荧光素标记的发夹引物作为供体，使用4-（4'-二甲基氨基苯基偶氮）苯甲酸（DABCYL）作为淬灭剂，获得35：1的信噪比。修饰的发夹引物不干扰DNA聚合酶的活性，并且可以使用热稳定的Pfu和Taq聚合酶。该方法用于检测前列腺特异性抗原的cDNA。结果表明，扩增产物的荧光强度与掺入引物的量相关，并且可以检测到少至10个分子的初始模板。该技术消除了残留污染的风险，简化了扩增测定，并为在极宽的动态范围内实时定量扩增的DNA开辟了新的可能性。

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