Volume 9, Issue 2 (May 2021)
Res Mol Med (RMM) 2021, 9(2): 81-102 |
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Asif S, Khan M, Arshad M W, Shabbir M I. PCR Optimization for Beginners: A Step by Step Guide. Res Mol Med (RMM) 2021; 9 (2) :81-102
URL: http://rmm.mazums.ac.ir/article-1-414-en.html
URL: http://rmm.mazums.ac.ir/article-1-414-en.html
1- Department of Biological Sciences, Faculty of Basic and Applied Sciences, International Islamic University, Islamabad, Pakistan.
2- Department of Molecular Biology, Faculty of Basic Medical Sciences, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan.
3- Department of Biological Sciences, Faculty of Basic and Applied Sciences, International Islamic University, Islamabad, Pakistan. , imran.shabbir@iiu.edu.pk
2- Department of Molecular Biology, Faculty of Basic Medical Sciences, Shaheed Zulfiqar Ali Bhutto Medical University, Islamabad, Pakistan.
3- Department of Biological Sciences, Faculty of Basic and Applied Sciences, International Islamic University, Islamabad, Pakistan. , imran.shabbir@iiu.edu.pk
Abstract: (4997 Views)
Background: The invention of Polymerase Chain Reaction (PCR) marked a breakthrough in biomedical research. Its invention divided the timeline into an era before and after PCR. Because of its multiple applications, it has become a vital tool for clinical researchers and diagnosticians. A effective PCR experiment requires adequate knowledge of each reaction component and step-by-step procedure to attain the optimized results. This research aims to employ optimization strategies that are easy to perform, cost-effective, and do not require PCR kits for the generation of amplicons for TYR, MITF, and SOX10 genes and can be used in sequence analysis.
Materials and Methods: Whole blood samples were used to extract genomic DNA with an inorganic method. DNA quantification was done by spectrophotometry analysis. Optimization strategies were adopted to generate PCR products of candidate genes and visualized by agarose gel electrophoresis. Sanger sequencing was performed to check the quality and specificity of generated amplicons via optimization strategies.
Results: This study demonstrates a novel approach for troubleshooting failed reactions without the use of PCR kits. The result indicated that use of sterilized material and reagents along with optimum MgCl2 concentration (2.0-3.0 mM), DNA quantity (25 ng/μL), and annealing temperature (54-60°C) are necessary to achieve successful amplification. Sequence analysis revealed no background noise often associated with sequencing results.
Conclusion: Optimizing MgCl2 concentration, DNA quantity, annealing temperature, along with the use of contamination-free material and reagents are essential steps in PCR optimization. Following this guide, anyone lacking proper supervision, and with little or no knowledge of the procedure should attain the desired results.
Materials and Methods: Whole blood samples were used to extract genomic DNA with an inorganic method. DNA quantification was done by spectrophotometry analysis. Optimization strategies were adopted to generate PCR products of candidate genes and visualized by agarose gel electrophoresis. Sanger sequencing was performed to check the quality and specificity of generated amplicons via optimization strategies.
Results: This study demonstrates a novel approach for troubleshooting failed reactions without the use of PCR kits. The result indicated that use of sterilized material and reagents along with optimum MgCl2 concentration (2.0-3.0 mM), DNA quantity (25 ng/μL), and annealing temperature (54-60°C) are necessary to achieve successful amplification. Sequence analysis revealed no background noise often associated with sequencing results.
Conclusion: Optimizing MgCl2 concentration, DNA quantity, annealing temperature, along with the use of contamination-free material and reagents are essential steps in PCR optimization. Following this guide, anyone lacking proper supervision, and with little or no knowledge of the procedure should attain the desired results.
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