新生隐球菌基因组DNA不同抽提方法的比较  

Comparative study of extraction methods for genomic DNA of Cryptococcus neoformans

作　　者：邹先彪[1,2] 廖万清[2] 温海[2] 吴建华[3] 仇芸[2] 杨金水[4] 

机构地区：[1]北京市解放军总医院304临床部皮肤科,北京100048 [2]第二军医大学长征医院皮肤科,上海200003 [3]第二军医大学长海医院皮肤科,上海200433 [4]复旦大学遗传所,上海200433

出　　处：《中国真菌学杂志》2010年第2期109-112,共4页Chinese Journal of Mycology

基　　金：国家自然科学基金(39770002)

摘　　要：目的 DNA是进行分子生物学研究的重要基础。在本研究中,我们建立了2种简单快速抽提基因组DNA的方法并可用作PCR扩增的模板。通过比较4种不同的DNA抽提方法以确定哪种更适合进行下一步的基因分析。方法这4种方法是:玻璃珠法,酶法,3%SDS法和氯化苄法。玻璃珠法是用玻璃珠在混漩器上剧烈振荡破碎细胞壁;3%SDS法是将细胞在含10mmol/LDTT的3%SDS溶液中加热,然后用5mmol/LKAc和异丙醇抽提,DNA的产量通过A260测定。结果 3%SDS溶解法、经典酶法、玻璃珠法和氯化苄法的DNA产量分别为0.4154±0.0367、0.8484±0.0756、1.2636±0.2040、0.4070±0.0339(g/L×108CFU/mL)。结论玻璃珠法是最敏感、重复性好、简单、费用合理的抽提方法 。Objective To establish simple and rapid methods for extracting genomic DNA from C.neoformans.To compare and search the better one for gene analysis.Methods Glass beads method,classical lytic enzyme treatment,3%SDS lysis and the benzyl chloride method were set up and compared.In glass beads method,cells were broken by vigorously mixing with glass beads on a votorex;In 3%SDS method,cells were heated in 3%SDS extraction buffer containing 10 mmol/L DTT for 20 minutes.The lysate was extracted with 5 mmol/L KAc and isopropanol.Yield of DNA was calculated from the A260 for clean DNA.Results DNA production by 3%SDS lysis,classical lytic enzyme treatment,glass beads method and the benzyl chloride method was 0.415 4±0.036 7,0.848 4±0.075 6,1.263 6±0.204 0,0.407 0±0.033 9 （g/L×10^8 CFU/mL） respectively.Conclusions Glass beads method was more sensitve,reproducible,simple and cost-effective for DNA extraction.

关 键 词：新生隐球菌 DNA抽提 方法 

分 类 号：R379.5[医药卫生—病原生物学]