REAL-TIME PCR: AN APPROPRIATE APPROACH TO CONFI RM SSDNA GENERATION FROM PCR PRODUCT IN SELEX PROCESS

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Kouhpayeh Shirin; HEJAZI ZAHRA; KHANAHMAD HOSSEIN; REZAEI ABBAS

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Journal Paper

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Journal: Iranian Journal of Biotechnology Year:2017 | Volume:15 | Issue:2 Page(s): 0-0

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Title

REAL-TIME PCR: AN APPROPRIATE APPROACH TO CONFI RM SSDNA GENERATION FROM PCR PRODUCT IN SELEX PROCESS

Author(s)

Kouhpayeh Shirin | HEJAZI ZAHRA | KHANAHMAD HOSSEIN | REZAEI ABBAS | Issue Writer Certificate

Keywords

HALF- RENATURATION

MELT CURVE

PAGE

REAL TIME PCR

SELEX

Abstract

Background: Aptamers are single stranded DNA (ssDNA) or RNA molecules. The potential of aptamers for binding to the diff erent 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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APA: Copy

Kouhpayeh, Shirin, HEJAZI, ZAHRA, KHANAHMAD, HOSSEIN, & REZAEI, ABBAS. (2017). REAL-TIME PCR: AN APPROPRIATE APPROACH TO CONFI RM SSDNA GENERATION FROM PCR PRODUCT IN SELEX PROCESS. IRANIAN JOURNAL OF BIOTECHNOLOGY, 15(2), 0-0. SID. https://sid.ir/paper/690362/en

Vancouver: Copy

Kouhpayeh Shirin, HEJAZI ZAHRA, KHANAHMAD HOSSEIN, REZAEI ABBAS. REAL-TIME PCR: AN APPROPRIATE APPROACH TO CONFI RM SSDNA GENERATION FROM PCR PRODUCT IN SELEX PROCESS. IRANIAN JOURNAL OF BIOTECHNOLOGY[Internet]. 2017;15(2):0-0. Available from: https://sid.ir/paper/690362/en

IEEE: Copy

Shirin Kouhpayeh, ZAHRA HEJAZI, HOSSEIN KHANAHMAD, and ABBAS REZAEI, “REAL-TIME PCR: AN APPROPRIATE APPROACH TO CONFI RM SSDNA GENERATION FROM PCR PRODUCT IN SELEX PROCESS,” IRANIAN JOURNAL OF BIOTECHNOLOGY, vol. 15, no. 2, pp. 0–0, 2017, [Online]. Available: https://sid.ir/paper/690362/en

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