合浦珠母贝DNA的抽提和RAPD反应体系的优化  

DNA extraction of the pearl oyster Pinctada fucata and optimization of RAPD conditions

作　　者：郭奕惠[1] 黄桂菊[1] 喻达辉[1] 

机构地区：[1]中国水产科学研究院南海水产研究所,广东广州510300

出　　处：《南方水产》2006年第4期59-64,共6页South China Fisheries Science

基　　金：广东省科技计划项目(2002B2150101);广东省自然科学基金项目(037148)

摘　　要：分别从保存于-70℃和95%乙醇的合浦珠母贝组织提取总DNA。结果表明,乙醇保存标本用双蒸馏水预处理后所提的DNA与-70℃保存的标本所提的DNA质量均较好。用抽提的DNA为模板优化RAPD条件,得到适合于合浦珠母贝的RAPDPCR反应条件为:20μL反应体系中包括20ngDNA模板,1×PCRBuffer,0.1mmol·L-1dNTPs,2.5mmol·L-1MgCl2,0.2μmol·L-1引物,1UTaqDNA聚合酶。最佳退火温度40℃。PCR产物用非变性PAGE银染和琼脂糖EB染色检测所得到的主要带型相同,但PAGE能检测到更多的多态带。Total DNA were extracted from the pearl oyster Pinctada fucata tissues preserved at -70℃ and fixed in 95% alcohol, respectively. The results showed that the quality of DNA extracted from alcohol preserved tissue through washing with distilled water was as good as that from frozen tissue. The condition for RAPD analysis was optimized and the appropriate reaction was as follows： a 20 μL reaction includes 20 ng template DNA, 1 × PCR buffer, 0. 1 mmol·L^-1 dNTPs, 2.5 mmol·L^-1 MgCl2 , 0. 2 μmol·L^-1 each primer, and 1 U Taq DNA polymerase. The optimal annealing temperature is 40℃. The PCR products were separated using non-denatured polyacrylamide gel electrophoresis （PAGE） with silver staining and agarose electrophoresis with ethidium bromide staining, respectively. Both PAGE and agarose separation presented consistent common bands but PAGE can reveal more polymorphic bands.

关 键 词：合浦珠母贝 DNA提取 RAPD条件优化 PAGE-银染 

分 类 号：Q781[生物学—分子生物学]