Real-Time PCR: an Appropriate Approach to Confirm ssDNA Generation from PCR Product in SELEX Process
Real-Time PCR: an Appropriate Approach to Confirm ssDNA Generation from PCR Product in SELEX Process
复制标题
DOI：
10.15171/ijb.1550
复制
发表时间：
2017-03-01
影响因子：
1.3
通讯作者：
Rezaei, Abbas
中科院分区：
文献类型：
--
作者：
Kouhpayeh, Shirin;Hejazi, Zahra;Rezaei, Abbas
背景：适体是单链DNA（ssDNA）或RNA分子。适体与不同靶点结合的潜力使其被广泛用作首选的诊断和治疗工具。DNA适体由于其更高的稳定性、更容易的选择和生产而呈现出优于RNA寡核苷酸的几个优点。通过指数富集配体系统进化（SELEX）方法筛选DNA适体在很大程度上取决于双链DNA（dsDNA）能否成功转化为ssDNA。虽然ssDNA的可视化仅限于基于凝胶的方法，但由于超过1015个不同的序列，该方法不适用于SELEX的初始轮次。本研究的目的是评估效率的另一种技术，用于确认的ssDNA的产生相比，聚丙烯酰胺电泳（PAGE）analysis.Materials和方法：实时PCR扩增的初始库，其次是酶切的dsDNA在本研究中。随后进行解链曲线分析以评价从dsDNA产生ssDNA。结果：熔解曲线显示，dsDNA转化为ssDNA的基础上，从73.8至41.5 ℃的Tm显着降低。PAGE分析不能有效地显示ssDNA从相应的初始dsDNA文库中产生的情况，但可以有效地根据SELEX轮数的增加来确认ssDNA的产生。本研究证明了真实的-时间PCR作为用于验证初始文库制备的DNA适体选择过程中ssDNA生成的合适确认技术。
Background: Aptamers are single stranded DNA (ssDNA) or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment (SELEX) method is much dependent on the successful conversion of double stranded DNA (dsDNA) to ssDNA.Objective: There are different methods available for ssDNA generation. While visualization of ssDNA is limited to the gel-based method, the method is not applicable in the initial rounds of SELEX due to more than 1015 different sequences. This study was designed to evaluate the efficiency of another technique for confirming the ssDNA generation in comparison to the polyacrylamide electrophoresis (PAGE) analysis.Materials and Methods: Real-time PCR was employed in the present study for PCR amplification of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis.Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a significant reduction of Tm from 73.8 to 41.5 degrees C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was efficient enough to confirm ssDNA generation in accordance with the increasing the number of SELEX rounds.Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation.