新生隐球菌基因组DNA不同抽提方法的比较

中国真菌学杂志　 2010, Vol. 5 　Issue (2): 109-112.

新生隐球菌基因组DNA不同抽提方法的比较

邹先彪^1,2, 廖万清², 温海², 吴建华³, 仇芸², 杨金水⁴

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

收稿日期:2010-02-11 出版日期:2010-04-28 发布日期:2010-04-28
作者简介:邹先彪,男(汉族),博士,副主任医师.E-mail:xbzou@126.com
基金资助:
国家自然科学基金(39770002)

Comparative study of extraction methods for genomic DNA of Cryptococcus neoformans

ZOU Xian-biao^1,2, LIAO Wan-qing², WEN Hai², WU Jian-hua³, QIU Yun², YANG Jin-shui ⁴

1. Department of Dermatology, 304th Hospitol Affiliated to PLA General Hospital, Beijing 100048, China;
2. Department of Dermatology, Changzheng Hospitol, The Second Military Medical University, Shanghai 200003, China;
3. Department of Dermatology, Changhai Hospitol, The Second Military Mcrlical Universtiy, Shanghai 200433, China;
4. Insititute Genetic of Fudan Universtiy,Shanghai 200433, China;

Received:2010-02-11 Online:2010-04-28 Published:2010-04-28

摘要/Abstract

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。

关键词: 新生隐球菌, DNA抽提, 方法

Abstract: 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×10⁸ CFU/mL) respectively.Conclusions Glass beads method was more sensitve,reproducible,simple and cost-effective for DNA extraction.

Key words: Cryptococcus neoforman, DNA extraction, methods

中图分类号:

R379.5

邹先彪, 廖万清, 温海, 吴建华, 仇芸, 杨金水. 新生隐球菌基因组DNA不同抽提方法的比较[J]. 中国真菌学杂志, 2010, 5(2): 109-112.

ZOU Xian-biao, LIAO Wan-qing, WEN Hai, WU Jian-hua, QIU Yun, YANG Jin-shui. Comparative study of extraction methods for genomic DNA of Cryptococcus neoformans[J]. Chinese Journal of Mycology, 2010, 5(2): 109-112.

参考文献

[1] Illnait-Zaragozi MT,Manínez-Machín GF,Fernández-Andreu CM,et al.Microsatellite typing of clinical and environmental Cryptococcus neoformans vat.grubii isolates from Cuba shows multiple genetic lineages[J].PLoS ONE,2010,5(2):e9124.
[2] 邹先彪,廖万清,温海,等.新生隐球菌原生质体形成条件的研究[J].第二军医大学学报,2001,22(11):22-24.
[3] Zhu H,Qu F,Zhu LH.Isolation of genomic DNAs from plants,fungi and bacteria using benzyl chloride[J].Nucleic Acids Research,1993,21 (22):5279-5280.
[4] 彭秀玲,袁汉英,谢毅,等.基因工程实验技术[M].第二版.长沙:湖南科学技术出版社,1998.
[5] Pericolini E,Cenci E,Monari C,et al.Cryptococcus neoformans capsular polysaccharide component galactoxylomannan induces apoptosis of human T-cells through activation of caspase-8[J].Cellular Microbiology,2006,8 (2):267-275.
[6] Zaragoza O,Telzak A,Bryan RA,et al.The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is rearranged during budding[J].Molecular Microbiology,2006,59(1):67-83.
[7] Charlier C,Chre'tien F,Baudrimont M,et al.Capsule structure changes associated with Cryptococcus neoformans crossing of the blood-brain barrier[J].Am J Pathol,2005,166(2):421-432.
[8] Perfect JR.Cryptococcus neofromans:a sugar-coated killer.In molecular principles of fungal pathogenesis[J].Heitman J,Filler SG,Edwards JE Jr,Mitchell AP,2006:281-303.ASM Press,Washington DC.
[9] Casadevall A,Perfect JR.Cryptococcus neoformans[M].ASM Press,Washington DC.1998.
[10] Sorrell-TC,Brownlee AG,Ruma P,et al.Concordance of clinical and environmental isolates d Cryptococcus neoformans var.gattii by amplification of polymorphic DNA analysis and PCR fingerprinting[J].J Clin Microbiol,1996,34 (5):1253 -1260.
[11] Rappelli P,Are R,Casu G,et al.Deveiopment d a nested PCR for detection of Cryptoeoccus neoformans in cerebrospinal fluid[J].J Clin Microbiol,1998,36(11):3438-3440.
[12] 吴建华,廖万清,柴建华.快速抽提新生隐球菌DNA的新方法[J].中华皮肤科杂志,1994,27(1):39-40.
[13] Wairt S,Stateva L,Palittapongarnpim P.C Loning and heterologous expression of Cryptocococcus neoformans CNSRB1 cDNA in Saccharomyces cerevisiae[J].Southeast Asian J Trop Med Public Health,2008,39(3):484-491.
[14] Sandhu GS,Kline BC,Stockman L,et al.Molecular probes for diagnosis of fungal infections[J].J Clin Micrpbiol,1995,33(11):2913-2919.
[15] Varma A,Kwon-Chung KJ.Rapid method to extract DNA from Cryptococcus neoformans[J].J Clin Microbiol,1992,29 (4):810-812.
[16] Sandhu GS,Kline BC,Stockman L,et al.Molecular probes for diagnosis of fungal infections[J].J Clin Microbiol,1995,33(11):2913-2919.