利用氯化苄提取适于分子生物学分析的真菌 DNA

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。

ISOLATION OF GENOMIC DNAS FROM FUNGI USING BENZYL CHLORIDE

With rapid progress of biotechnology, the difficulty to isolate high quality DNAs becomes a main obstacle. Almost all of the reported fungus DNAs extraction protocols include homogenizing in liquid nitrogen or digested with enzymes to destroy cell walls and membrances. Thus, these treatments are time consuming and tedious. Benzyl chloride is a chemical reagent which, under weak alkalic conditon, can react with hydroxyl residues in polysaccharides, such as chitin, thus destroy the sugar chain of fungal cell walls. Genomic DNAs are then released with the least mechanic shearing. Here, with the use of benzyl chloride, a simple, fast and inexpensive procedure for fungal genomic DNA preparation is developed. With this method, the fungal genomic DNAs are extracted with stable yields and little contamination of proteins or other organic components. The isolated DNAs are suitable for digestion with restriction endonucleases, RFLP analysis, PCR amplification and other molecular biological experiments. No report concerning DNA preparation with benzyl chloride has been read ever before in national or international publications.