作者：宋君1,3, 张容2, 宋昭彬3, 郭灵安1, 王东1, 雷绍荣1
作者单位：1. 四川省农业科学院分析测试中心, 四川成都 610066;
2. 中国医学科学院输血研究所, 四川成都 610000;
3. 四川大学生命科学学院, 四川成都 610064
关键词：AFLP; 基因组DNA制备; 条件优化; 银染
摘要：
AFLP(选择性扩增片断长度多态性)指纹技术被认为是当今最有效的DNA指纹分析技术之一,但是由于该技术操作流程较长,对模板DNA质量要求较高以及PCR反应成分等变数条件的影响,要获得高清晰的DNA指纹图谱比较困难。文中以齐口裂腹鱼为研究对象,对AFLP的分析条件作了一定的探索。结果表明:基因组DNA必须是高纯度、高分子量;酶切充分;PCR反应最佳条件为:引物E-XYZ,M-XYZ在体系中的浓度分别为1ng/mL,5ng/mL;dNTPs浓度为0.2mmol/L;MgCl2浓度为1.9mmol/L。银染时在NaOH条件下显色比在传统银染法中的Na2CO3条件下显色,图谱背景更干净,条带更清晰。
Discussion on analyzing DNA fingerprint of schizothorax prenanti with AFLP
SONG Jun1,3, ZHANG Rong2, SONG Zhao-bin3, GUO Ling-an1, WANG Dong1, LEI Shao-rong1
1. Center of Analysis and Testing, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China;
2. Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu 610000, China;
3. School of life Sciences, Sichuan University, Chengdu 610064, China
Abstract: AFLP (Amplified Fragment Length Polymorphism) fingerprinting was regarded as one of the most useful techniques in nowadays for DNA fingerprint analysis, but sometimes it was difficult to obtain a perfect DNA fingerprint graph for many factors such as much longer operating processes for this technology, more high quality requirements for DNA templates, more variable for PCR ingredients and so on. Schizothorax prenanti was regarded as the research focus of this article, and the optimum AFLP condition was suggested. The results indicated that genome DNA must be high purification and high molecular weight, and DNA template must be fully digested if perfect DNA fingerprint graphs were expected. The optimum reaction conditions for PCR: the concentrations for EXYZ and M-XYZ in system were 1 ng/ml and 5 ng/ml respectively, and those for dNTPs and MgCl2 were 0.2 mmol/L, and 1.9 mmol/L respectively. If the sliver-stain processed in NaOH other than in Na2CO3, much clearer DNA fingerprint bands and much cleaner background of fingerprint graph could be obtained.
Keywords: AFLP; Genome DNA preparation; Condition optimization; Sliver-stain
2008, 34(6): 125-127 收稿日期: 2007-7-25;收到修改稿日期: 2007-9-21
基金项目:
作者简介: 宋君(1973-),男,四川成都市人,助理研究员,博士研究生,主要从事转基因生物检测技术的研究。
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