分子信标
核酸外切酶 III
化学
核酸外切酶
寡核苷酸
DNA
核酸酶
检出限
杂交探针
低聚物限制
荧光
分子生物学
组合化学
基因
色谱法
生物化学
DNA聚合酶
生物
光学
物理
大肠杆菌
作者
Xiaolei Zuo,Fan Xia,Yi Xiao,Kevin W. Plaxco
摘要
A limitation of many traditional approaches to the detection of specific oligonucleotide sequences, such as molecular beacons, is that each target strand hybridizes with (and thus activates) only a single copy of the relevant probe sequence. This 1:1 hybridization ratio limits the gain of most approaches and thus their sensitivity. Here we demonstrate a nuclease-amplified DNA detection scheme in which exonuclease III is used to "recycle" target molecules, thus leading to greatly improved sensitivity relative to, for example, traditional molecular beacons without any significant restriction in the choice of target sequences. The exonuclease-amplified assay can detect target DNA at concentrations as low as 10 pM when performed at 37 degrees C, which represents a significant improvement over the equivalent molecular beacon alone. Moreover, at 4 degrees C we can obtain a detection limit as low as 20 aM, albeit at the cost of a 24 h incubation period. Finally, our assay can be easily interrogated with the naked eye and is thus amenable to deployment in the developing world, where fluorometric detection is more problematic.
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