Real-Time PCR: an Appropriate Approach to Confi rm ssDNA Generation from PCR Product in SELEX Process

Document Type: Research Paper

Authors

1 Department of Immunology, Isfahan University of Medical Sciences, Isfahan, Iran.

2 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 313, Iran

3 Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable disease, Isfahan University of Medical Sciences, Isfahan, 313, Iran

4 Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 313, Iran

Abstract

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 diff erent methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased
method, the method is not applicable in the initial rounds of SELEX due to more than 1015 diff erent sequences. This
study was designed to evaluate the effi ciency of another technique for confi rming 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 amplifi cation 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 signifi cant reduction of Tm from 73.8 to 41.5 °C. Applying PAGE analysis, it was not eff ectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was effi cient enough to confi rm 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 confi rmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation.

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