实验免RAPD反应体系的优化及引物筛选

The Optimization of RAPD Reaction System and Primers Screening in Experimental Rabbit

目的建立实验兔随机扩增多态DNA（RAPD）的最优反应体系，并对RAPD引物进行筛选优化。方法以白毛黑眼（WHBE）兔，日本大耳白（JW）兔，新西兰（NZW）兔为实验材料，对影响RAPD反应的各因素进行优化，摸索出最佳的Mg^2＋，dNTP，Taq酶，引物和DNA模板的浓度，并从60个随机引物中筛选出适合实验兔RAPD反应的引物。结果最优RAPD—PCR反应体系为：在20山的反应体系中，Mg^2＋1．5mmol／L，dNTP0．25mmol／L，Taq酶1．25U，引物5μmol／L，模板DNA40ng。60个RAPD引物中剔除了重复性差，条带模糊的引物后，有25个引物稳定，扩增出来的条带清晰、多态性高。结论本研究筛选出的25个引物和建立的PCR体系可应用于实验兔的RAPD反应。

Objective The optimal reaction system and primers of RAPD-PCR in experimental Rabbits were established. Methods One hundred and fifty samples were collected from WHBE rabbits, JW rabbits and NZW rabbits, and their DNA was extracted by standard procedure of phenol/chloroform. The optimization of its RAPD-PCR reaction system, the most appropriate concentration of Mg^2＋, dNTP, Taq polymerase, random primers and DNA template, was studied. Sixty primers were used to amplify DNA of experimental rabbits with RAPD-PCR methods. Results The optimal PCR system for RAPD analysis was carried out in 20 μl reaction volumes containing the following： 10xloading buffer 2 μl, Mg^2＋1.5 mmol/L, dNTP 0.25 mmol/L, Taq polymerase 1.25 Units, random primer 5 μmol/L, DNA template 40 ng and sterile distilled water to total volume. On the basis of preliminary experiments, twenty-five primers with high polymorphism were selected in our study. Conclusion The selected 25 primers and optimalizing PCR reaction system can applied to RAPD reaction in experimental rabbit.