用Chelex-100方法从牦牛乳中提取总DNA扩增线粒体DNA序列

Extraction of total DNA from yak milk using Chelex-100 method for the amplification of mitochondrial DNA sequences

旨在建立一种从牦牛乳中快速提取总DNA并扩增线粒体基因的方法.实验以20头麦洼牦牛的乳为试验材料,用Chelex-100方法提取总DNA,扩增线粒体DNA(mtDNA)的D-loop区和细胞色素b基因(Cytb).结果显示,Chelex-100法能利用20μL全乳或脱脂乳快速制备总DNA,用于mtDNA的D-loop区和Cytb基因的PCR扩增,条带清晰并能直接测序.该方法的PCR扩增一般需要45个循环,扩增效果存在一定的个体差异,且全乳优于脱脂乳,半奶牦牛乳优于全奶牦牛乳.建立的方法既可使用全乳,也可采用脱脂乳大规模提取总DNA,并扩增线粒体基因用于后续研究.

This study aimed to establish a method for rapid extraction of total DNA from yak milk and amplification of mitochondrial genes. Milk samples of 20 Maiwa yaks were used,and total DNA was extracted using the Chelex-100 method followed by the amplification of the mitochondrial DNA（ mtDNA） D-loop region and the cytochrome b gene（ Cytb）. The results showed that the Chelex-100 method could use 20 μL of whole milk or skim milk to prepare total DNA rapidly for PCR amplification of the D-loop region and Cytb gene. The PCR bands were clear and could be directly sequenced. The present PCR amplification generally required 45 cycles,and there existed differences in amplification efficiency among sample individuals; The whole milk was better than the skim milk,and the milk of half-lactating yak was better than that of the total-lactating yak. The method established in this study can use both whole milk and skim milk for large-scale extraction of total DNA in order to amplify the mitochondrial genes for subsequent studies.