Chen Jing, Liu Xiaohui, Xu Meng, Li Zhoumin, Xu Danke
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Jinling College, Nanjing University, Nanjing 210089, China.
Biomicrofluidics. 2021 Apr 5;15(2):024107. doi: 10.1063/5.0045965. eCollection 2021 Mar.
One of the main obstacles for systematic evolution of ligands by exponential enrichment (SELEX) failure is the generation of a non-specific product, as selection-inherent amplification procedures tend to form by-products, which prevents the enrichment of target-binding aptamers. Herein, we reported a dual-microfluidic amplified system (dual-MAS) based on the real-time polymerase chain reaction (PCR) detection chip and the large volume PCR chip for one-step specific PCR and for evaluating the SELEX process. First, it is a simple method to accomplish analytical PCR and amplification PCR in one step, and the optimal number of cycles for generating the specific PCR product is the cycles when the slope of the linear amplification period of the real-time PCR curve begins to decrease. Second, the time used by the dual-MAS for generating a specific PCR product is reduced to 30 min, and the multi-functional dual-MAS can simultaneously evaluate the SELEX process by providing important information on the amounts of enriched sequences and the library diversity in every round of SELEX. In addition, pollution contamination and fragment loss can be significantly avoided in the closed chip. Last, the specific PCR product, the amounts of enriched sequences, and the library diversity can be obtained for every single SELEX in just 30 min. Compared with current methods, this system can reduce the time for generating a specific PCR product and SELEX, and it is easier to choose the optimal number of cycles for a specific PCR product. In a word, it is a sensitive, simple, and rapid strategy to improve the specificity of the PCR product and make the process of SELEX in a controlled way.
指数富集配体系统进化技术（SELEX）失败的主要障碍之一是产生非特异性产物，因为基于选择的固有扩增程序往往会形成副产物，这会阻止靶标结合适体的富集。在此，我们报道了一种基于实时聚合酶链反应（PCR）检测芯片和大容量PCR芯片的双微流控扩增系统（dual-MAS），用于一步特异性PCR和评估SELEX过程。首先，这是一种在一步中完成分析PCR和扩增PCR的简单方法，产生特异性PCR产物的最佳循环数是实时PCR曲线线性扩增期斜率开始下降时的循环数。其次，dual-MAS产生特异性PCR产物的时间缩短至30分钟，多功能dual-MAS可以通过提供每轮SELEX中富集序列数量和文库多样性的重要信息来同时评估SELEX过程。此外，在封闭芯片中可显著避免污染和片段损失。最后，只需30分钟就能为每一次单独的SELEX获得特异性PCR产物、富集序列数量和文库多样性。与现有方法相比，该系统可以减少产生特异性PCR产物和进行SELEX的时间，并且更容易选择特异性PCR产物的最佳循环数。总之，这是一种提高PCR产物特异性并以可控方式进行SELEX过程的灵敏、简单且快速的策略。
