鲮鱼DAF和DNA谱带的快速银染检测  

DNA amplification fingerprinting of mud carp (Cirrhina molitorella) and fast silver staining of DNA profiles

作　　者：郑光明[1] 朱新平[1] 刘毅辉[1] 罗建仁[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所 中国水产科学研究院热带亚热带鱼类选育与养殖重点实验室,广东广州510380

出　　处：《中国水产科学》2005年第3期336-339,共4页Journal of Fishery Sciences of China

基　　金：国家自然科学面上基金(30271022);广东省自然科学基金(001159).

摘　　要：利用20个高GC含量的8bp的随机引物开展野生鲮鱼(Cirrhinamolitorella)DNA扩增指纹的研究,以建立鲮鱼DNA扩增指纹(DAF)技术,包括引物设计,PCR反应条件的设置。结果显示,DAF引物浓度相对较高,最佳引物浓度为3 5μmol/L。DAF引物pn 16(GCGACCTG)扩增表明,DAF有好的重复性,引物pn-16扩增特征性谱带可用于鉴定鲮鱼。DAF扩增产物可揭示鲮DNA的多态性。同时建立了DNA尿素-聚丙烯酰胺凝胶银染快速检测技术和最佳银染参数,银染检测大约需要1h,建立包括固定、漂洗、染色、显色、停显色的银染程序和尿素-聚丙烯酰胺凝胶湿法与干法的保存方法。DNA amplification fingerprinting of genome DNA of wild mud carp(Cirrhina molitorella)were obtained using 20 8-mer primers with high GC content .The DNA amplification fingerprinting technique was established,including primer device,and PCR reaction condition.In the condition,primer concentration was relatively high,and 3.5μmol/L was the maximum.DAF had good repetition using primer pn-16(GCGACCTG);the characteristic DNA amplification profiles of Primer pn-16 were identified in mud carp.DNA amplification fingerprinting of some primers revealed DNA polymorphic.Mean while,fast silver staining of DNA profiles in urea-polyacrylamide gel was applied in DNA amplification fingerprinting.The best silver staining condition parameters was obtained,including the silver concentration and length of AgNO_(3) impregnation,the effects of formaldehyde and thiosulfate concentration,and the imaging development temperature.The concentration of AgNO_(3) 1g/L and impregnation of 30min could get optimal sensitivity.The optimal formaldehyde(37%-40%) concentration was 150μL/L.2mg/L Na_(2)S_(2)O_(3) was optimal concentration for reducing nonspecific background staining.The silver staining technique was also applied to the identification of mud carp by RAPD Primer.The silver staining procedure included fixing,rinsing,impregating,developing and stopping.The method of wet and dry preserved gels was established.

关 键 词：鲮 DNA扩增指纹 银染 

分 类 号：Q959.46[生物学—动物学]