Ejaz M, Gaisheng Z, Na N, Huiyan Z, Qidi Z, Qunzhu W
College of Agronomy, Northwest A&F University, Yangling, Shaanxi, China.
College of Agronomy, Northwest A&F University, Yangling, Shaanxi, China
Genet Mol Res. 2014 Dec 4;13(4):10320-31. doi: 10.4238/2014.December.4.27.
We evaluated and compared 2 mitochondrial DNA (mtDNA) extraction methods in terms of DNA quality and success of subsequent polymerase chain reaction (PCR) amplifications from yellow etiolated shoots of wheat crop (Triticum aestivum). mtDNA ex-traction is difficult because the presence of metabolites interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The method (with modification) involved inactivation of genomic DNA by DNase I enzyme, RNA by RNase enzyme, contaminant proteins by using proteinase K, and precipitation of polysaccharides in the presence of a high salt concentration. The DNase I and RNA enzyme ratio was adjusted to 10:8 mL. The purity of mtDNA was confirmed by PCR amplification of genomic, mitochondrial, and chloroplast (rbcL) gene. The mitochondrial COXIII gene of 400 bp was amplified; the b-actin and chloroplast genes were not amplified. A260/A280 (1.89) and A260/A230 (2.07) ratios were calculated using a spectrophotometer. The isolated mtDNA was amenable to amplification and restriction digestion. The technique is fast, reproducible, and suitable for PCR-based markers.
我们评估并比较了两种线粒体DNA（mtDNA）提取方法，从黄化小麦幼苗（普通小麦）中提取的DNA质量以及后续聚合酶链反应（PCR）扩增的成功率。mtDNA提取困难，因为代谢物的存在会干扰DNA分离程序以及诸如DNA限制性内切酶、扩增和克隆等下游应用。该方法（经过改进）包括用脱氧核糖核酸酶I使基因组DNA失活，用核糖核酸酶使RNA失活，用蛋白酶K去除污染蛋白，并在高盐浓度下沉淀多糖。将脱氧核糖核酸酶I和核糖核酸酶的比例调整为10:8 mL。通过对基因组、线粒体和叶绿体（rbcL）基因进行PCR扩增来确认mtDNA的纯度。扩增出了400 bp的线粒体COXIII基因；β-肌动蛋白和叶绿体基因未被扩增。使用分光光度计计算A260/A280（1.89）和A260/A230（2.07）的比值。分离出的mtDNA适合进行扩增和限制性内切酶消化。该技术快速、可重复，适用于基于PCR的标记。
