一管酒精法提取转基因小鼠胚胎组织基因组的实验方法及应用

Protocol and application of ethanol method to extract genomic DNA from transgenic mouse embryos

摘要:

目的：优化提取基因组的方法，建立高效、简便、快速提取基因组DNA的方法，用于大批量转基因小鼠鉴定。方法：用一管酒精基因组DNA抽提法抽提基因组DNA。结果：经过分光光度法检测基因组DNA浓度、纯度及电泳分析DNA质量，显示一管酒精抽提法提取的基因组与经典酚氯仿提取法提取的基因组产量、纯度及质量没有明显差别；酶切全基因组后，2种方法获得的基因组都可以被酶完全切开；两者模板都能成功应用PCR扩增来鉴定基因敲入小鼠并可应用于基因组测序及Real-time PCR检测基因敲入小鼠中外源基因并鉴定其相对拷贝数。结论：一管酒精基因组抽提法得到的DNA质量可靠，可高效、简便、快速鉴定转基因小鼠及进行基因组DNA酶切、测序、Real-time PCR等后续实验。

Abstract:

Objective:To optimize the method of genome extraction and to establish an effective,convenient and fast research platform which could identify and screen the transgenic mice on a large scale.Methods:The Genomic DNA was extracted by using the ethanol method.Results:The results of spectrophotometry and gel electrophoresis revealed that the concentration,purity and quality of the genomic DNA extracted through the ethanol method had no obvious difference compared with that extracted through the classical phenolchloroform method.The genomic DNA obtained by the two extraction protocols could both be digested completely and able to identify the knock-in mice by PCR amplification.Both the templates were well used in gene sequencing and Real-time PCR to detect the exogenous gene and the relative copy numbers. Conclusion:The DNA extracted through the ethanol method was superior in quality and had stable and reliable test results in transgenic mice identifying,digestion,gene sequencing and Real-time PCR amplification.