家畜冷冻精液DNA的纯化及影响因素分析

DNA purification from livestock frozen semen and analysis of influencing factors

[目的]基于人精液DNA试剂盒提取法,优化家畜冷冻精液DNA纯化方法,探讨冷冻精液中影响DNA提取质量的因素。[方法]使用摩拉水牛冷冻精液,按100μL(或200μL)样品初始量,以及0.5、1、1.5、2、3和5 h的蛋白酶解时间,用试剂盒法纯化DNA。从DNA产物浓度、A 260/A 280和A 260/A 230值3方面,结合琼脂糖凝胶电泳和PCR检测进行分析,最终确定DNA纯化的最佳精液初始量以及最佳蛋白质酶解时间。在此基础上,纯化67份来源于牛、马、驴等不同家畜品种的冷冻精液DNA,同时测定每份精液中蛋白质、甘油/甘油三酯和果糖的含量,结合精液样品的冻存时间,研究以上4种因素对DNA纯化质量的影响。[结果]使用100μL冷冻精液,蛋白酶解3 h的纯化效果最好,而且酶解2和3 h的DNA产物,均可以满足后续核基因组和线粒体基因组常规PCR的要求。家畜冷冻精液中蛋白质、甘油/甘油三酯和果糖含量与提取DNA浓度、A 260/A 280和A 260/A 230值(纯度)在不同程度上呈斯皮尔曼负相关,其中果糖是干扰DNA纯化的关键因素,同时样品的冻存时间与DNA质量存在显著正相关关系。虽然冷冻精液中蛋白质、甘油/甘油三酯和果糖等成分可以减轻冷冻复苏对精子的损伤,但这些成分的增加,会对DNA纯化质量产生一定的负面影响,即使它们随着冻存时间及DNA纯化时酶解时间的延长而有所消耗。[结论]优化后的人精液DNA试剂盒法可完全适用于家畜冷冻精液DNA的纯化。

[Objectives]This study aimed to optimize the method to purify DNA from livestock frozen semen using a DNA kit designed for human semen,and to explore the components influencing the quality of DNA extraction in frozen semen.[Methods]Frozen semen DNA from Murrah buffalo was extracted with a kit,according to 100μL(or 200μL)of starting sample volume as well as 0.5,1,1.5,2,3 and 5 h protease incubation time.DNA products were analyzed by concentration,A 260/A 280 and A 260/A 230 in combination with agarose gel electrophoresis and PCR amplification to determine the best starting volume and protease digestion time for DNA purification.Then frozen semen from 67 different bovines,horses and donkeys breeds were accordingly used for DNA purification.The amounts of protein,glycerol/triglyceride and fructose were measured,and frozen time was recorded simultaneously for each sample to investigate the relationships between these factors and DNA products.[Results]The results showed that DNA purification starting with 100μL frozen semen after 3 h protease digestion reproducibly provided the best quality of DNA,and longer protease incubation showed no significant improvement.Both the DNA products at 2 and 3 h could be used for subsequent regular nuclear genome and mitochondrial genome studies.In addition,the amounts of protein,glycerol/triglyceride and fructose from livestock frozen semen were negatively correlated with the concentration as well as A 260/A 280 and A 260/A 230 purities of extracted DNA to different extents by a Spearman’s correlation analysis.Among these factors,fructose was a key factor that interfered with DNA purification.There was also a significant positive correlation between frozen time and DNA quality.Although protein,glycerol/triglyceride,and fructose prevent sperm from damage during freezing and thawing,elevating the amount of these components could have a negative impact on DNA purification,even if these factors could be ameliorated by prolongation of frozen time and digestion time during DNA purification.[Conclusions]The human semen DNA kit method can be fully applied to purifying DNA from livestock frozen semen after optimization.