扩展青霉基因组DNA五种提取方法比较  

Comparison of Five Different Methods for Extracting Genomic DNA from Penicillium expansum

作　　者：李亚菲[1] 何鸿举[1] 吕茜[1] 樊明涛[1] 

机构地区：[1]西北农林科技大学食品科学与工程学院,陕西杨凌712100

出　　处：《北方园艺》2011年第20期128-131,共4页Northern Horticulture

基　　金：教育部博士点基金资助项目(200807120017);陕西省科技攻关资助项目(2007K01-12)

摘　　要：为寻找一种适合于扩展青霉基因组DNA的提取方法,比较了固体培养、液体静置培养和液体振荡培养3种培养方式的提取效果,分别采用氯化苄法、蜗牛酶法、CTAB法、SDS法及异硫氰酸胍法提取扩展青霉菌基因组DNA,经DNA质量浓度测定及电泳分析,比较5种方法的差异,并利用扩展青霉的多聚半乳糖醛酸酶(Polygalacturonase)基因内一段保守序列,设计PCR引物,扩增大小为288bp的特异目的基因,检测其灵敏性。结果表明:固体培养的方式较适合扩展青霉基因组DNA的提取,另外5种提取方法均能提取出扩展青霉菌基因组DNA,扩增出目的条带,其中氯化苄法和蜗牛酶法所提DNA的纯度均较好,但蜗牛酶法所提DNA量较少。因此,氯化苄法是5种提取方法中最可靠的DNA提取方法,所提DNA浓度高、纯度好、结果稳定,可作为PCR反应的模板进行特异性扩增,且该方法的最低检测限大约为1.01×10-1μg/mL。The aim was to compare DNA extraction methods and find one that was suitable for genomic DNA extraction from Penicillium expansum. The effect of extraction in the three culture ways including the solid, liquid stalling and liquid oscillation was compared. Genomic DNA were extracted using five methods including benzyl chloride, snail enzyme, SDS, CTAB, guanidinium isothiocyanate. The effects of the different extraction methods were evaluated through the quality determination and PCR analysis which was based on conservative sequence of the polygalacturonase gene of the Penicillium expansum to amplify a length of 288 bp, testing its sensitivity. The results showed that solid cultivation approach was suitable for the extraction of genomic DNA from Penicilliurn expansum, all five extraction methods could extract the genomic DNA to amplify the target DNA, of which benzyl chloride and snail enzyme methods were good with snail enzyme being a little less DNA. Therefore, the benzyl chloride method was the best choice to extract DNA from Penicillium expansum with high concentration and purity to carry out specific PCR,and the limit of detecting DNA was 0. 1 ug/mL.

关 键 词：扩展青霉菌 DNA提取 氯化苄法 蜗牛酶法 SDS法 咖法 异硫氰酸胍法 

分 类 号：Q939.1[生物学—微生物学]