Advanced Photonics Research Institute, Gwangju Institute of Science and Technology, 261 Chemdan-gwagiro, Gwangju 500-712, Republic of Korea.
Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology, 261 Chemdan-gwagiro, Gwangju 500-712, Republic of Korea.
Analyst. 2018 Feb 7;143(3):695-699. doi: 10.1039/c7an01520h. Epub 2018 Jan 4.
We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and HO at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.
我们开发了一种新的策略，用于通过利用在 5'末端用抗 DNAzyme 序列修饰的靶特异性引物来对 PCR 产物进行比色检测。单链 DNAzyme 序列折叠成具有血红素的 G-四链体结构，并表现出很强的过氧化物酶活性。当互补链与 DNAzyme 序列结合时，它阻止 G-四链体结构的形成并失去其过氧化物酶活性。在存在靶基因的情况下，PCR 扩增进行，并且在反应混合物中存在的抗 DNAzyme 序列修饰引物通过引物延伸形成双链。因此，它不会阻止 DNAzyme 序列。此外，通过在反应结束时添加 2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)(ABTS)和 HO，产生比色信号。我们已经成功地在缓冲液中检测到单个 HIV-1 gag 基因拷贝和 10 个人血清拷贝。所开发的策略可以通过简单的引物设计来检测复杂生物样品中的 DNA 和 RNA，该设计包括 DNAzyme 和延伸的 DNA 引物。
