基于DNA提取方法优化转基因小鼠基因分型研究

Optimization of DNA extraction method for genotyping in transgenic mice

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100 μL 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 moreover，the DNA lysis buffer preparation was simple and convenient，enabling more reaction times，and reducing reagent costs and workload considerably.Conclusion This study provides an economical，simple，and reliable DNA extraction technique for genotyping in transgenic mice，which deserves application and promotion.