<br>双功能 Phi29 DNA 聚合酶触发指数滚环扩增，用于对长链基因组 DNA 中嵌入的目标 DNA 进行序列特异性检测。,Scientific Reports

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双功能 Phi29 DNA 聚合酶触发指数滚环扩增，用于对长链基因组 DNA 中嵌入的目标 DNA 进行序列特异性检测。
Scientific Reports ( IF 3.8 ) Pub Date : 2017-07-24 , DOI: 10.1038/s41598-017-06594-1
Xiao-Yu Li _{1,

2} , Yi-Chen Du ₂ , Yu-Peng Zhang ₂ , De-Ming Kong _{1,

2,

3}

Affiliation

开发了一种核酸外切消化辅助指数滚环扩增 (RCA) 策略，用于对长链基因组 DNA 中嵌入的目标 DNA 进行灵敏且序列特异性的检测。在此，Phi29 DNA聚合酶发挥核酸外切酶和聚合酶两个重要作用。长链基因组 DNA 经超声处理后可断裂成小 DNA 片段。含有目标 DNA 的片段与线性挂锁探针杂交，触发环状 RCA 模板的形成。从目标 DNA 序列 3' 端伸出的尾部随后会被 Phi29 DNA 聚合酶的 3' → 5' 核酸外切酶活性消化，即使它们折叠成双链结构。消化后的 DNA 片段可以启动后续的 RCA 反应。 RCA 产物被设计为折叠成 G 四链体结构，当适当的切口核酸内切酶识别位点合理地引入 RCA 模板时，RCA 产物会呈指数累积。该方法被证明适用于使用人类病原体新型隐球菌作为模型进行真实基因组 DNA 检测。此外，这项工作还有另外两个重要发现：首先，3'尾的存在可以保护RCA引物不被Phi29 DNA聚合酶降解。其次，Phi29 DNA 聚合酶的 3' → 5' 核酸外切活性适用于单链和双链 DNA。

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Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA.

An exonucleolytic digestion-assisted exponential rolling circle amplification (RCA) strategy was developed for sensitive and sequence-specific detection of target DNA embedded in long-stranded genomic DNA. Herein, Phi29 DNA polymerase plays two important roles as exonuclease and polymerase. Long-stranded genomic DNAs can be broken into small DNA fragments after ultrasonication. The fragments that contain target DNA, hybridize with a linear padlock probe to trigger the formation of a circular RCA template. The tails protruding from the 3'-end of the target DNA sequences are then digested by the 3' → 5' exonuclease activity of Phi29 DNA polymerase even if they fold into a double-stranded structure. The digested DNA fragments can then initiate subsequent RCA reaction. RCA products, which are designed to fold into G-quadruplex structures, exponentially accumulate when appropriate nicking endonuclease recognition sites are introduced rationally into the RCA template. This method is demonstrated to work well for real genomic DNA detection using human pathogen Cryptococcus neoformans as a model. In addition, this work has two other important discoveries: First, the presence of a 3'-tail can protect the RCA primer from degradation by Phi29 DNA polymerase. Second, 3' → 5' exonucleolytic activity of Phi29 DNA polymerase can work for both single- and double-stranded DNA.

更新日期：2017-07-25

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