Repository of Research and Investigative Information

Repository of Research and Investigative Information

Baqiyatallah University of Medical Sciences

Essential strategies to optimize asymmetric PCR conditions as a reliable method to generate large amount of ssDNA aptamers

(2017) Essential strategies to optimize asymmetric PCR conditions as a reliable method to generate large amount of ssDNA aptamers. Biotechnology and Applied Biochemistry. pp. 541-548. ISSN 0885-4513

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Abstract

Asymmetric PCR, a simple method to generate single-stranded DNA (ssDNA) aptamers in systematic evaluation of ligand by exponential enrichments rounds, is coupled with limitations. We investigated the essential strategies for optimization of conditions to perform a high-quality asymmetric PCR. Final concentrations of primers and template, the number of PCR cycles, and annealing temperature were selected as optimizing variables. The qualities of visualized PCR products were analyzed by ImageJ software. The highest proportion of interested DNA than unwanted products was considered as optimum conditions. Results revealed that the best values for primers ratio, final template concentration, annealing temperature, and PCR cycles were, respectively, 30:1, 1 ng/L, 55 degrees C, and 20 cycles for the first and 50:1, 2 ng/L, 59 degrees C, and 20 cycles for other rounds. No significant difference was found between optimized asymmetric PCR results in the rounds of two to eight (P > 0.05). The ssDNA quality in round 10 was significantly better than other rounds (P < 0.05). Generally, the ssDNA product with less dimers, double-stranded DNA (dsDNA), and smear are preferable. The dsDNA contamination is the worst, because it can act as antidote and inhibits aptameric performance. Therefore, to choose the best conditions, the lower amount of dsDNA is more important than other unwanted products. (C) 2016 International Union of Biochemistry and Molecular Biology, Inc.

Item Type: Article
Keywords: asymmetric PCR optimization ssDNA aptamer single-stranded-dna polymerase-chain-reaction therapeutics selex Biochemistry & Molecular Biology Biotechnology & Applied Microbiology
Divisions:
Page Range: pp. 541-548
Journal or Publication Title: Biotechnology and Applied Biochemistry
Journal Index: ISI
Volume: 64
Number: 4
Identification Number: https://doi.org/10.1002/bab.1507
ISSN: 0885-4513
Depositing User: مهندس مهدی شریفی
URI: http://eprints.bmsu.ac.ir/id/eprint/4348

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