放大器
故障排除
聚合酶链反应
桑格测序
DNA提取
基因组DNA
计算生物学
琼脂糖
计算机科学
DNA
色谱法
DNA测序
生物
化学
基因
遗传学
操作系统
作者
Saaim Asif,NULL AUTHOR_ID,Maaz Khan,Muhammad Arshad,Muhammad Imran Shabbir,NULL AUTHOR_ID,NULL AUTHOR_ID,NULL AUTHOR_ID
标识
DOI:10.32598/rmm.9.2.1189.1
摘要
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 stepby- 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.
科研通智能强力驱动
Strongly Powered by AbleSci AI