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检测植物DNA扩增多态性方法的比较和改进
引用本文:胡志昂,恽锐.检测植物DNA扩增多态性方法的比较和改进[J].Acta Botanica Sinica,1997,39(2):144-148.
作者姓名:胡志昂  恽锐
作者单位:中国科学院植物研究所,中国科学院植物研究所,中国科学院植物研究所,中国科学院植物研究所,中国科学院植物研究所,中国科学院植物研究所 北京 100093,北京 100093,北京 100093,北京 100093 美国俄亥俄州Miami大学植物学和化学系。Present address:Department of Botany and Chemistry,Miami University,Oxford,Ohio 45056 USA.,北京 100093,北京 100093
基金项目:国家科委攀登项目,中国科学院重大和重点项目
摘    要:以辽东栎(Quercus liaotungensis Koidz.)、锦鸡儿(Cargagana ssp.)和野大豆(Glycine soja(L.)Sieb.etZucc.)为材料,比较了随机扩增多态DNA(RAPD)和DNA扩增指纹(DAF)方法。用RAPD的琼脂糖胶电泳和溴乙锭染色,RAPD和DAF谱一般不足10条带。用DAF的变性聚丙烯酰胺凝胶电泳(PAGE)和银染,极大地提高了RAPD的灵敏度和分辨率,多达20~40个产物。用3'末端完全相同的引物,RAPD和DAF有同样的扩增谱,说明两种方法有相似的机理。降低胶的浓度可提高RAPD和DAF的分辨率,达40~80条带。琼脂糖电泳分离的溴乙锭显示的单荧光带,用PAGE和银染可分辨出多个片段。分子克隆证实单荧光带的分子量异质性。在用Taq DNA多聚酶的条件下,RAPD和DAF的再现性均良好。

关 键 词:随机扩增多态DNA  DNA扩增指纹  植物自然群体  DNA多样性  聚丙烯酰胺凝胶电泳  银染

COMPARISON AND IMPROVEMENT OF POLYMORPHISM AMPLIFICATION METHODS USED FOR DETECTING DNA DIVERSITY OF PLANTS
Authors:Hu Zhi-ang  Yun Rui  Zhong Min  Dong Fu-gui  Wang Hong-xin and Qian Ying-qian
Abstract:A comparative study on randomly amplified polymorphic DNA (RAPD) and DNA amplification fingerprinting (DAF) used in detecting DNA diversity of natural populations of liaodong oak ( Quercus liaotungensis Koidz.), peashrub ( Caragana ssp.) and wild soybean ( dycine soja (L.) Sieb. et Zucc.) was conducted. In case of using agarose gel electrophoresis with ethidium bromide (EB) staining which was traditionally used in RAPD, both methods generally showed simple profiles with less than 10 bands. By using urea-polyacrylamide gel electrophoresis (Urea-PAGE) with silver staining which was usually used in DAF, the resulted RAPD profiles, similar to those in DAF ones, became much more resolved and consisted of 20 to 40 bands. Similar profile was shared by RAPD and DAF when primer OPA-02 (TACCGAGCTG) and 7.7a (CGAGCTG) were included respectively. It indicated that both methods may have similar mechanism of amplification. After running a-garose gel electrophoresis and EB staining the individual bands were cut off and rerun in Urea-PAGE with silver staining. It showed that each EB-stained band consists of several fragments with different length. Gene cloning confirmed that a single EB-stained band in agarose gel did contain many DNA fragments with different molecular weight. Low experimental variability has been found in both RAPD and DAF even if Taq DNA polymerase was used. An improved method of RAPD with PAGE and silver staining has been successfully used in detection of DNA diversity for natural populations of plants.
Keywords:Random amplified polymorphic DNA  DNA amplification fingerprinting  Plant natural population  DNA diversity  Polyacrylamide gel electrophoresis  Silver staining  
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