Optimization of DNA extraction method for genotyping in transgenic mice
夏雨 1余静 1康露 1张欣 2马娓 1严军1
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作者信息
1. 陆军军医大学大坪医院野战外科研究部、创伤与化学中毒全国重点实验室,重庆 400042
2. 重庆医科大学附属第二医院康复医学科,重庆 400010
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摘要
目的:探讨DNA提取方法优化后对转基因小鼠基因分型结果的影响,并比较分析与业内常用试剂盒基因型鉴定结果的差异.方法:在团队前期专利基础上,改进了 DNA裂解液的配方和实验流程,并以新型转录因子Musculin和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因小鼠为例,对基因分型结果进行了比较研究和验证.结果:DNA提取时现配裂解液配方为100μL 0.025 N NaOH工作液、160 μL 0.5 M EDTA工作液和40 mL超纯水;每个样本加入180 μL裂解液,100 ℃ 30 min.与相关领域常用的基因分型试剂盒相比,该DNA提取优化方案能够在确保鉴定准确的前提下有效缩短基因分型时间,而且裂解液配置方法简单,反应次数多,步骤更简便,可大幅降低试剂成本和工作量.结论:本研究提供1种适用于转基因小鼠基因分型的经济、简单、可靠的DNA提取技术,具有较好的应用和推广价值.
Abstract
Objective:To explore the influence of optimized DNA extraction on genotyping of transgenic mice,and to compare it with commonly used reagent kits in the field of scientific research.Methods:Based on our previous patent,we modified the DNA lysis buffer formula and experimental protocol.Genotyping and validation were carried out using musculin(a novel transcription factor)-transgenic mice and enhanced green fluorescent protein-transgenic mice.Results:The DNA lysis solution was prepared immediately before use,and was composed of 100 µL 0.025 N NaOH,160 μL 0.5 M EDTA,and 40 mL ultrapure water.Each sample was mixed with 180 μL DNA lysis solution,at 100 ℃ for 30 min.Compared with the commonly used genotyping kits in relevant fields,the optimized DNA extraction method effectively shortened the genotyping time while ensuring the accuracy of identification,and more-over,the DNA lysis buffer preparation was simple and convenient,enabling more reaction times,and reducing reagent costs and work-load considerably.Conclusion:This study provides an economical,simple,and reliable DNA extraction technique for genotyping in transgenic mice,which deserves application and promotion.
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