Neo Jia Ling, Uttamchandani Mahesh
Department of Chemistry, Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore, Singapore.
Methods Mol Biol. 2013;1039:141-51. doi: 10.1007/978-1-62703-535-4_12.
We describe a method to detect DNA sequences visually through a color change reaction using DNAzymes. We successfully applied the assay for the detection of Salmonella and Mycobacterium DNA, as well as for genotyping single base differences from within human genomic DNA samples. Our approach adopts a split probe targeting system, designed with G-rich sequences, which reassembles in the presence of target DNA, producing G-quadruplexes with catalytic activity. Asymmetric PCR is first performed to amplify the target region into single-stranded copies, with primer ratios tailored for optimum amplification. This is followed by direct addition of the visual probes, substrates, and reagents to produce a color change within 15 min should the desired target sequences be present. This approach hence offers a rapid readout, ease-of-use, and handling convenience, especially at the point-of-care.
我们描述了一种通过使用DNA酶的颜色变化反应来直观检测DNA序列的方法。我们成功地将该检测方法应用于沙门氏菌和分枝杆菌DNA的检测，以及对人类基因组DNA样本中的单碱基差异进行基因分型。我们的方法采用了一种分裂探针靶向系统，该系统设计有富含G的序列，在目标DNA存在的情况下重新组装，产生具有催化活性的G-四链体。首先进行不对称PCR，将目标区域扩增为单链拷贝，通过调整引物比例以实现最佳扩增。随后直接加入视觉探针、底物和试剂，如果存在所需的目标序列，15分钟内就会产生颜色变化。因此，这种方法提供了快速读数、易用性和操作便利性，特别是在即时检测点。
