人类精液DNA提取方法的探讨及影响因素分析

Discussion on Extraction of Human Semen DNA and Analysis of Its Influencing Factors

目的建立一种最优化的提取高质量人类精液基因组DNA的方法,为辅助生殖技术男性不育分子生物学科学研究提供技术支持。方法提取人类精液基因组DNA,优化设计实验步骤,研究初始样品类型、初始样品量、二硫苏糖醇(DTT)剂量、蛋白酶K(PK)消化时间、混匀震荡等因素对DNA的提取效果的影响,应用核酸定量和琼脂糖凝胶电泳分析比较各组提取效果的差异。结果应用自然分层后的精液沉淀提取DNA较原始精清可获得较高浓度的DNA。初始样品量为100～150μl时可获得较好的提取效果,随着样本量的增加,DNA提取效果逐渐下降。在样本中加入DTT可以明显提高DNA产量,DTT的加入利于DNA裂解及提取,且增加了DNA的稳定性。PK适宜消化时间为5～8 h;时间过短(<2 h),DNA裂解不全提取效果较差,时间过长(>12 h)则会造成DNA降解。在PK消化时,摇床震荡可以提高DNA产量。结论应用自然分层后的精液沉淀提取DNA,当初始样品量为100～150μl时,在样本中加入1M DTT 20μl,PK消化时间为5～8 h,并在消化时摇床震荡,可以获得高质量的基因组DNA。

Objective To establish an optimal extraction method of high-quality human semen genomic DNA,which can provide the necessary technical support for molecular biology science of male infertility in assisted reproductive technology. Methods To extract human semen genomic DNA,we optimized the design of the experimental procedure ,and studied the factors that might have an impact on the effects of DNA extraction ,such as the initial sample type,the amount of initial sample,DTT dose,protease K（PK） digestion time,mix concussion and others.Nucleic acid quantification and agarose gel electrophoresis were used to compare the extraction effects among all groups .Results Compared with the supernatant group,the natural-stratification precipitated semen group got a higher concentration of DNA .The initial sample volume of 100 -150 μl obtained better extraction results;DNA extraction effect decreased gradually with increasing sample volume.The addition of DTT in the sample improved the yield of DNA significantly ,and the added DTT promoted DNA cleavage and extraction as well as increased the stability of DNA .The appropriate PK digestion time ranged from 5 hours to 8 hours;The short PK digestion time（〈2 hours） led to less effective extraction for incomplete DNA cleavage,and the long PK digestion time（〉12 hours） caused the degradation of DNA.The mix concussion could increase the yield of DNA while PK digestion worked.Conclusion With the initial sample volume of 100-150μl,1M DTT of 20μl added,PK digestion time of 5-8 hours,and mix concussion is performed while PK digestion works ,high-quality genomic DNA can be obtained from the natural-stratification precipitated semen .