【摘要】 以扬子鳄总DNA为材料,对影响SRAP-PCR的Mg2+、dNTPs、TaqDNA聚合酶、引物浓度等因素进行了优化,分析了TaqDNA聚合酶、样本浓度、Mg2+浓度、dNTPs浓度以及引物浓度对SRAP-PCR扩增结果的影响.筛选出扩增条带清晰、多态性丰富的SRAP引物l3对,建立了稳定的、可重复的扬子鳄SRAP-PCR最佳反应体系及PCR扩增参数.研究结果表明:在30μlSRAP-PCR反应体系中,样本最适宜为1μl(30ng/μl),Mg2+的最适为2μl(2.5mmol/L),dNTPs最适为2μl(2.5mmol/L),单引物的最适均为1μl(10pmol/μl);聚合酶在30μl反应体系中宜加入1U.SRAP-PCR引物筛选及其反应体系的建立,为今后利用SRAP标记技术开展扬子鳄种群的分子生物学研究提供了一个标准化程序和强有力的工具.更多还原

【Abstract】 Chinese alligator(Alligator sinensis)is a critically endangered endemic species under legislative protection.Selecting suitable SRAP-PCR primers and establishing the optimal reaction system is the base of Alligator wild populations and captive population genetic research.The concentrations of Mg2+,dNTPs,Taq DNA polymerase,primers which affect the SRAP-PCR reactions were optimized in order to establish the SRAP molecular marker system in Chinese Alligator.The effects of primer pairs,whole DNA concentration,Mg2+ concentration,Taq DNA polymerase concentration and dNTPs concentration on SRAP reaction system were studied.Altogether 13 primer pairs were selected which can amplify unequivocally visible,monomorphic bands.And we accessed a stable,repeatable SRAP-PCR reaction system and the best of PCR parameters.The results showed that in 30μl PCR reaction: template DNA 1μl(30ng/μl),Mg2+(2.5mmol/L)2μl,dNTPs(2.5mmol/L)2μl,each primer(10pmol/μl)1μl;Taq DNA polymerase 1 U,The work provides a standardized procedure and powerful tool for molecular biology research of the Chinese Alligator.更多还原