荷包猪RAPD-PCR反应条件研究

[目的]建立适合荷包猪RAPD-PCR反应的最佳反应体系。[方法]以荷包猪为试验材料,以MgCl2浓度、引物浓度、dNTP浓度、模板DNA用量、TaqDNA聚合酶用量及退火温度为影响因子,在保持其他影响因子一致的条件下,变化单一因子,筛选最优参数,研究各影响因子对RAPD-PCR反应的影响。[结果]最佳反应体系的总体积为20.0μl,MgCl2浓度为2.5μmol/L、引物浓度2.0μmol/L、dNTP浓度400.0μmol/L、模板DNA为100 ng/μl、TaqDNA聚合酶为1.0 U。PCR反应程序:94℃预变性2 min(94℃变性1 min,36℃退火1 min,72℃延伸1 min,循环40次),72℃延伸5 min。此反应体系所扩增出来的结果比较稳定,带型清晰且亮度适中。[结论]该研究为应用RAPD技术对荷包猪作进一步的遗传分析奠定了基础。

Study on the RAPD-PCR Reaction Conditions for Hebao Pig

[Objective] The aim was to establish the optimum reaction system suitable for Hebao pig RAPD-PCR reaction.[Method] With Hebao pig as experimental material,Mg2+ concn.,primers concn.,dNTPs concn.,template DNA amount,Taq DNA polymerase amount and anneal temperature as influential factors,single factor was changed under the condition of keeping other influential factors consistent,the optimum parameter was screened and the effect of each influential factor on RAPD-PCR reaction were studied.[Result] The total volume of the optimum RAPD system was 20.0 μl,including 2.5 μmol/L MgCl2,2.0 μmol/L primers,400.0 μmol/L dNTP,100 ng/μl template DNA and 1.0 U Taq DNA polymerase.The PCR reaction program was as follows: pre-denaturalization at 94 ℃ for 2 min,followed by 40 cycles,each cycle including denaturalization at 94 ℃ for 1 min,anneal at 36 ℃ for 1 min,extension at 72 ℃ for 1 min,and a final extension at 72 ℃ for 5 min.The amplified result under the reaction system was more stable,the band was clear and the band brightness was moderate.[Conclusion] The research laid the foundation for further genetic analysis of Hebao pig by using RAPD technology.