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Mohammadinezhad R, Jalali SAH, Farahmand H. Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences. Anal Methods. 2020;12(30):3823-3835doi: 10.1039/d0ay00491j.
Mohammadinezhad, R., Jalali, S. A. H., & Farahmand, H. (2020). Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences. Analytical methods : advancing methods and applications, 12(30), 3823-3835. https://doi.org/10.1039/d0ay00491j
Mohammadinezhad, Rezvan, et al. "Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences." Analytical methods : advancing methods and applications vol. 12,30 (2020): 3823-3835. doi: https://doi.org/10.1039/d0ay00491j
Mohammadinezhad R, Jalali SAH, Farahmand H. Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences. Anal Methods. 2020 Aug 14;12(30):3823-3835. doi: 10.1039/d0ay00491j. Epub 2020 Jul 17. PMID: 32676627.
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Anal Methods. 2020 Aug 14;12(30):3823-3835. doi: 10.1039/d0ay00491j. Epub 2020 Jul 17.

Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences.

Analytical methods : advancing methods and applications

Rezvan Mohammadinezhad, Seyed Amir Hossein Jalali, Hamid Farahmand

Affiliations

  1. Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran. [email protected].

PMID: 32676627 DOI: 10.1039/d0ay00491j

Abstract

Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an iterative method for in vitro selection of aptamers from a random synthetic oligonucleotide library. Successful retrieving of aptamers by SELEX relies on optimization of various steps including target immobilization, aptamer partitioning, amplification, and ssDNA generation, which all require spending considerable effort and cost. Furthermore, due to the random nature of the initial library, SELEX may redirect toward the selection of low-affinity aptamers that are over-represented in the ssDNA population due to PCR bias. Thus, precise monitoring of the SELEX process is crucial to ensure the selection of target-specific aptamers. In the present study, we investigated the reliability and simplicity of different direct and indirect monitoring methods including UV-Vis spectroscopy, real-time PCR quantification and melt-curve analysis, electrophoretic mobility shift assay (EMSA) and enzyme-linked oligonucleotide assay (ELONA) for selection of DNA aptamers for a protein target. All the examined methods were capable of illustrating the gradual evolution of specific aptamers by the progression of SELEX and showed almost similar results regarding the identification of the enriched round of selection. Moreover, we describe the use of melt-curve analysis in the colony real-time PCR method as a simple, robust, and repeatable tool for pre-sequencing separation of distinct aptamer clones.

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