利用氯化苄提取适于分子生物学分析的真菌 DNAISOLATION OF GENOMIC DNAS FROM FUNGI USING BENZYL CHLORIDE
朱衡,瞿峰,朱立煌
摘要(Abstract):
随着生物技术的飞速发展,更加需要简便、快速地提取高质量的DNA。以往报导的提取真菌DNA的方法大都采用液氮研磨或酶解破坏细胞壁和膜的方式,从而导致繁琐、复杂和费时的提取过程。根据氯化苄在弱碱条件下与多糖上的羟基反应形成醚从而使多糖长链断裂的事实,我们发展了一种全新的真菌DNA提取方法,该方法使氯化苄在pH9.0时与细胞壁多糖作用,破坏细胞壁,基因组DNA因而得以从细胞中释放出来。新方法具有简便、快速、价廉的优点,得到的DNA蛋白质污染少、质量较高、产量稳定。对该法提取的DNA作进一步的分子生物学分析,如限制性内切酶酶解、RFLP分析、RAPD扩增,都取得了令人满意的结果。利用氯化苄提取真菌DNA的研究,迄今在国际、国内均尚未见报道。
关键词(KeyWords): DNA提取;细胞壁;氯化苄
基金项目(Foundation): 863生物领域第一主题资助
作者(Author): 朱衡,瞿峰,朱立煌
DOI: 10.13346/j.mycosystema.1994.01.006
参考文献(References):
- [1] Boeke JD, Garfinkel DJ, Styles CA et al, 1985. Cell, 40:491-500
- [2] Timberlake WE, 1986. in Biology and Molecular Biology of Plant-Pathogen Interactions, NATO ASI Series H: Cell Biology, ed.Bailey, JA(Springer,Berlin) Vol 1:343-357
- [3] Hamer JE, Givan S,1990. Genetic mapping with dispersed repeated squences in the rice blast fungus: mapping the SMO locus. Mol Gen Genet, 223:287-495
- [4] 邢其毅,徐瑞秋,周政编《基础有机化学》(下册):p.1099-1100。
- [5] Williams JGK, Kubelik AR, Livak KJ et al, 1990. Nucleic Acids Research, 18:6531-6535
- [6] Zhu H, Jiang W, Wang J et al, 1992. DNA .fingerprinting and pathotype characterization of Pyricularia oryzea Isolates. Mycosystema, 5:89-98
- [7] Kresovich S, Williams JGK, McFerson JR et al, 1992. Characterization of genetic identifies and relationships of Brassica oleracca L. via a random amplified polymorphic DNA assay. Theor Appl Genet, 85:190-196
- [8] Sobral BWS, Honeycutt RJ, 1993. Hight output gentic mapping of polyploids using PGR-gene-rated markers.Theor Appl Genet,86:105-112
- [9] Zhu H, Qu F, Zhu, L. 1993. Isolation of genomic DNAs from plants fungi and bacteria using Benzyl Chloride: A novel procedure with extra ordinary simplicity (Submitted to Nucleic Acids Research)