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

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10μg基因组DNA于30μl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50V电压条件下4℃电泳20h，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20min，转膜采用20×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。

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

Objective To explore if ECL (E nhanced ChemiLuminescence)directnucleic acid labelling and detection systems ca n be used to t he fingerprinting of pig genomic DNA.Methods Southern blot hybridization was conducted using ECL direct nucleic acid labelling and det ection systems to Banna minipig inbred lines with HRP(Horseradish Peroxidase) la belled human origin probe α-globin 3’HVR.Results We acq uired the following available DNA transfer and hybridization conditions.In brie f，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 electroph oresised on 0.6% agarose gels at 50V in 1×TAE running buffer until all fragmen ts below 2 kilobase(kb) had run off the gel.Depurination，denaturation and neut ralization were performed for 15min，15min and 20min respectively and the DNA w as transferred onto the positively charged nylon membrane for 1h in vacuum trans fer unit，hybridized in tube at 42℃ for 12h with probe 3'HVR labelled by HRP，a utoradiographied for 30min at room temperature，the clearly DNA fingerprint was achieved.Conclusion This me thod can be used to DNA fingerprinting of pigs and the same effect can be achiev ed at that generated from the isotopically labelled probe.