化学发光加强法核酸标记检测系统用于猪DNA指纹分析的研究  

DNA Fingerprinting in Minipig with ECL Direct Nucleic Acid Labelling and Detection Systems

作　　者：陈丙波[1] 周建华[1] 魏泓[1] 曾养志[2] 

机构地区：[1]第三军医大学实验动物中心,重庆400038 [2]云南农业大学遗传资源研究室,昆明650201

出　　处：《中国实验动物学杂志》2001年第1期15-18,共4页Chinese Journal of Laboratory Animal Science

基　　金：国家重点基础研究发展规划项目! (G2 0 0 0 0 1 61 0 6) ;国家"九五"科技攻关项目! (96- 2 3- 0 6- 0 1 ) ;国家自然科学基金

摘　　要：目的　探讨化学发光加强法 (ECL)核酸直接标记和检测系统应用于猪的DNA指纹分析的可行性。方法　采用人源α -珠蛋白 3’HVR探针 ,应用ECL核酸直接标记和检测系统 ,对西双版纳小耳猪进行了Southern杂交DNA指纹分析 ,获得了最适实验条件。结果　取 10 μg基因组DNA于 30 μl反应体系中 ,加入HinfⅠ或HaeⅢ内切酶 3～ 5U/ μgDNA ,酶切消化 4h ,消化液在 5 0V电压条件下 4℃电泳 2 0h ,采用真空转移法进行脱嘌呤、碱变性、中和及转膜 ,时间分别为 15min、 15min及 2 0min ,转膜采用 2 0×SSC真空转移 1h至hybondN +尼龙膜上 ,大大提高了转膜效率。以HRP标记探针进行管中杂交 ,以化学发光法进行检测 ,室温放射自显影 30min ,获得了清晰可辨的DNA指纹图。结论　该方法可用于猪的DNA指纹分析 ,其效果与同位素标记探针的效果基本一致。Objective To explore if ECL (Enhanced ChemiLuminescence)direct nucleic acid labelling and detection systems can be used to the fingerprinting of pig genomic DNA.Methods Southern blot hybridization was conducted using ECL direct nucleic acid labelling and detection systems to Banna minipig inbred lines with HRP(Horseradish Peroxidase) labelled human origin probe α-globin 3’HVR.Results We acquired the following available DNA transfer and hybridization conditions.In brief,10μg genomic DNA was digested with 40u HinfⅠ or HaeⅢ endonuclease in 30μl total reaction volume for 4h at 37℃.After digestion,the sample was electrophoresised on 0.6% agarose gels at 50V in 1×TAE running buffer until all fragments below 2 kilobase(kb) had run off the gel.Depurination,denaturation and neutralization were performed for 15min,15min and 20min respectively and the DNA was transferred onto the positively charged nylon membrane for 1h in vacuum transfer unit,hybridized in tube at 42℃ for 12h with probe 3'HVR labelled by HRP,autoradiographied for 30min at room temperature,the clearly DNA fingerprint was achieved.Conclusion This method can be used to DNA fingerprinting of pigs and the same effect can be achieved at that generated from the isotopically labelled probe.

关 键 词：核酸 标记 探针 珠蛋白 放射自显影 嘌呤 DNA指纹分析 G基因 压条 杂交 

分 类 号：Q78[生物学—分子生物学] S435[农业科学—农业昆虫与害虫防治]