CAJ | 학술논문

采用不同的解冻方法以及牛奶体细胞分离与 SDS 苯酚法相结合的手段，分离提取－80℃冻藏牛奶中牛基因组 DNA，利用超微量核酸分析仪和琼脂糖凝胶电泳法分别对 DNA 浓度、纯度及分子量大小进行检测，采用 PCR 技术扩增牛特异性基因序列片段进行 DNA 质量鉴定。结果表明，“两步法”解冻提取的牛基因组 DNA 的质量优于“一步法”和“三步法”，且该方法所提取的 DNA 浓度为（52．46±2．40）μg／mL，DNA 纯度为1．85±0．80，条带较清晰；PCR 扩增证实这些 DNA 可以成功扩增出729 bp 的牛特异性基因序列片段。本研究成功优化了牛奶中 DNA 的分离提取方法，筛选出了冻藏牛奶的适宜解冻方法，建立了一种新的超低温冻藏牛奶中牛基因组 DNA 提取方法。
채용불동적해동방법이급우내체세포분리여 SDS 분분법상결합적수단，분리제취－80℃동장우내중우기인조 DNA，이용초미량핵산분석의화경지당응효전영법분별대 DNA 농도、순도급분자량대소진행검측，채용 PCR 기술확증우특이성기인서렬편단진행 DNA 질량감정。결과표명，“량보법”해동제취적우기인조 DNA 적질량우우“일보법”화“삼보법”，차해방법소제취적 DNA 농도위（52．46±2．40）μg／mL，DNA 순도위1．85±0．80，조대교청석；PCR 확증증실저사 DNA 가이성공확증출729 bp 적우특이성기인서렬편단。본연구성공우화료우내중 DNA 적분리제취방법，사선출료동장우내적괄의해동방법，건립료일충신적초저온동장우내중우기인조 DNA 제취방법。
Three thawing methods,milk somatic cell separation technique and SDS phenol method were used to separate and extract bovine genomic DNA from milk samples preserved at -80℃. The concentration and purity of the extracted DNA were measured by the ultra low volume spec-trometer,and molecular weight of DNA was obtained by agarose gel electrophoresis.The DNA quality was further identified by PCR to amplify specific bovine gene fragment.The results showed that DNA obtained from the two-step thawing method was better in quality than that from the one-step thawing method and three-step thawing method.The concentration and purity of the DNA from the two-step method were (52.46±2.40)μg/mL and 1.85±0.80,respective-ly.The specific bovine gene fragment (729 bp)could be successfully amplified,and the strip on gel image was clear.This study established a novel bovine genomic DNA extraction method from milk preserved at ultra-low temperature,by optimizing method for thawing frozen milk.