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

Iranian Journal of Biotechnology
Shirin KouhpayehAbbas Rezaei

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 different 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 different 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....Continue Reading

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Citations

May 22, 2019·Journal of Cellular Biochemistry·Shirin KouhpayehHossein Khanahmad
Jan 6, 2021·Molecular Biology Reports·Mina MirianHossein Khanahmad

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