Optimization and comparison of extraction methods of mitochondrial DNA of Oncomelania hupensis

作者：WANG Kang; XU Shu jun; WANG Yi xiao; CHEN Wen jie; WANG Yi nan; YU Fen fang; WANG Shao sheng; SUN En tao; ZHOU Shu lin

来源：中国血吸虫病防治杂志, 2017, 29(2).

DOI：10.16250/j.32.1374.2016228

• Oncomelania hupensis
• Mitochondrial genome DNA(mtDNA)
• Improved high-concentration-salt precipitation method

Objective To optimize the extraction methods of mitochondrial genome DNA(mtDNA)of Oncomelania hupen-sis. Methods The pyrolysis,protein K variable-temperature digestion and high-concentration potassium acetate purification were applied to optimize the high-concentration-salt precipitation method,and then the optimized method was compared with two common extraction methods,the sucrose density gradient centrifugation method and traditional high-concentration-salt pre-cipitation method. The mtDNA samples were identified by using spectrophotometry,agarose gel electrophoresis and the amplifi-cation products of COX1. The nuclear DNA contamination was tested by the amplification products of ITS. Results The concen-tration and yield of the improved method was significantly higher than those of the traditional method(F=3032.65,10185.00, both P <0.01). The mtDNA samples extracted were essentially free of nuclear DNA and protein,meeting PCR,sequence analy-sis and other molecular biology research requirements. Conclusions The improved high-concentration-salt precipitation meth-od for isolating mtDNA is simple,and it has high yield and low cost. The extracted mtDNA can meet relevant analysis require-ments.

• 单位
  皖南医学院