水牛全血基因组抽提方法优化

Optimization of Methods for Genomic Extraction of Buffalo Blood

[目的]通过比较三种提取水牛全血基因组DNA的方法,获得一种安全、高效、快速的DNA提取方法,为水牛基因功能的研究奠定基础。[方法]收集43头水牛抗凝血样,每份血样随机分为等量3份,每份血样2mL,分别使用试剂盒改良法、试剂盒说明书法及酚仿抽提法提取全血基因组DNA。比较分析三种方法提取的同一头牛三份血样DNA的含量、OD260/280值、琼脂糖凝胶电泳和基因扩增结果。[结果]三种方法提取DNA的含量由高到低依次为试剂盒改良法、酚仿抽提法、试剂盒说明书法,各组间差异显著(1376.72±127.54VS 1121.32±81.64VS 326.18±21.17,P<0.05)。三种方法提取DNA的OD-260/280值依次增加,试剂盒改良法与试剂盒说明书法OD260/280值差异不显著(1.85±0.0017 VS1.86±0.0021,P>0.05),与酚仿抽提法OD260/280值有显著差异(1.85±0.0017VS 1.88±0.0029,1.86±0.0021VS 1.88±0.0029,P<0.05)。三种方法提取DNA琼脂糖凝胶电泳结果显示,试剂盒改良法和试剂盒说明书法DNA条带清晰、光密度高,酚仿抽提法条带模糊有拖带现象。三种方法所提取的基因组PCR扩增stat5基因获得较清晰准确的条带,扩增效果良好。[结论]试剂盒改良法用于水牛全血基因组的提取比试剂盒说明书法和酚氯仿法更安全、高效、便捷。

【Objective】The aim of this study was to obtain an security,efficient and rapid DNA extraction method by comparing three genomic DNA extraction methods of buffalo,which would lay the foundation for gene function in buffalo.【Method】In this study,a total of 43 anticoagulant blood samples of buffalo were collected.Eachblood samples were randomly divided into three equal parts,and the volume of every part is 2 mL.Modified blood DNA Kit method,Blood DNA Kit instruction method and phenolchloroform extraction method were used to extract the genomic DNA from blood sample of same individual,respectively.Then,several contents,including the content of DNA,OD260/280,agar sugar gel rubber electrophoresis and gene amplification,were compared by using these three extraction methods.【Result】A significantly difference was found in the content of DNA by using these three extraction methods,and the order from high to low was the modified Blood DNA Kit method,phenol extraction and Blood DNA Kit instruction method（1376.72 ± 127.54 VS 1121.32 ± 81.64 VS 326.18 ± 21.17,P〈0.05）.Moreover,comparing the value of OD260/280,it was added by order,and there was not significant difference between modified Blood DNA Kit method and Blood DNA Kit instruction method（1.85±0.0017 VS 1.86± 0.0021,P〉0.05）,but it was significant differences among the phenol-chloroform extraction and other two groups（1.85±0.0017 VS 1.88±0.0029,1.86±0.0021 VS 1.88±0.0029,P 0.05）.Interestingly,the results of agarose gel electrophoresis by using these three DNA extraction groups showed that the DNA strap of modified Blood DNA Kit method and Blood DNA Kit instruction method was clear and high opticaldensity,but the DNA strap of phenol extraction method was fuzzy and towing.By amplifing stat5 gene,the results of three methods showed clear and accurate straps,and had good amplification effect consistently.【Conclusion】Compared with the Blood DNA Kit instruction method and phenol-chloroform extraction method,modified Blood DNA Kit method is a safer,more efficient and more convenient method for genomic extraction of buffalo blood.