采用AFLP分析齐口裂腹鱼DNA指纹的技术探讨

Discussion on analyzing DNA fingerprint of schizothorax prenanti with AFLP

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。

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 E- XYZ 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.