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Iranian Journal of Biotechnology، جلد ۱۵، شماره ۲، صفحات ۱۴۳-۱۴۸
عنوان فارسی
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کلیدواژه‌های فارسی مقاله
عنوان انگلیسی Real-Time PCR: an Appropriate Approach to Confirm ssDNA Generation from PCR Product in SELEX Process
چکیده انگلیسی مقاله 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 gel-based 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 efficiency of another technique for confirming 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 amplification 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 significant reduction of Tm from 73.8 to 41.5 °C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was efficient enough to confirm 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 confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation.  
کلیدواژه‌های انگلیسی مقاله Half- Renaturation, Melt Curve, PAGE, Real Time PCR, SELEX
نویسندگان مقاله شیرین کوهپایه |
department of immunology, school of medicine, isfahan university of medical sciences, isfahan, 313, iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

زهرا حجازی |
department of genetics and molecular biology, school of medicine, isfahan university of medical sciences, isfahan, 313, iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

حسین خان احمد |
department of genetics and molecular biology, school of medicine, isfahan university of medical sciences, isfahan, 313, iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

عباس رضایی |
department of immunology, school of medicine, isfahan university of medical sciences, isfahan, 313, iran
سازمان اصلی تایید شده: دانشگاه علوم پزشکی اصفهان (Isfahan university of medical sciences)

نشانی اینترنتی http://www.ijbiotech.com/article_49554_9d0e3f714824ea26f5b7f3523f762130.pdf
فایل مقاله اشکال در دسترسی به فایل - ./files/site1/rds_journals/441/article-441-485013.pdf
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