一种用于PCR的丝状真菌DNA快速粗提方法

A Rapid Alkaline Extraction Method for the Isolation of Crude Genomic DNA from Filamentous Fungi for PCR

为了在实验室条件下更加快速、经济、安全、高效地提取丝状真菌DNA,研究出了一种利用NaOH碱裂解快速提取DNA的方法,主要包括以下步骤:利用液氮快速粉碎样品;采用碱裂解(NaOH)的方式一步提取DNA;以TE缓冲液中和;以中和后的DNA作为模板进行基因克隆及PCR分析。通过比较不同的NaOH浓度提取尖孢镰刀菌DNA质量,确定最适NaOH浓度为0.5 mol/L。试验用此方法提取了丝状真菌尖孢镰刀菌(Fusarium oxysporum)、灰葡萄孢(Botrytis cinerea)、链格孢(Alternaria alternata)及核盘菌(Sclerotinia sclerotiorum)的基因组并用于基因克隆。同时,利用该方法提取的DNA质量可满足尖孢镰刀菌转化子的快速PCR鉴定需求。综上所述,与利用CTAB(十六烷基三甲基溴化铵)提取DNA相比,利用NaOH碱裂解法,可以更加快速提取丝状真菌的DNA,操作步骤简单,节约时间,无须使用氯仿、异丙醇等有毒物质,而且提取的丝状真菌DNA完整性较好,质量可观,可用于基因克隆及转化子筛选鉴定,具有一定的利用价值和推广前景。

In order to extract filamentous fungi DNA more quickly,economically,safely and efficiently under laboratory conditions,a rapid alkaline method for the direct extraction of DNA was developed including the following steps Grind the fungus tissues using liquid nitrogen;Adding alkaline lysis buffer(NaOH)to quickly extract DNA;Neutralizing extracted DNA sample with TE buffer;Performing PCR with neutralized DNA as template.It was found that DNA could be extracted with several NaOH concentrations,thereinto,0.5 mol/L NaOH was the recommended choice for the genomic DNA extraction from Fusarium oxysporum.Furthermore,we used this method to extract genomic DNA from filamentous fungi F.oxysporum,Botrytis cinerea,Alternaria alternata and Sclerotinia sclerotiorum for gene cloning.The method as described above was applicable to F.oxysporum transformants identification via PCR.Overall,compared to using CTAB(cetyl trimethyl ammonium bromide)for DNA extraction,NaOH could be used to isolate genomic DNA from filamentous fungi more quickly,and the entire extraction process was simple and time-saving.Specifically,toxic reagents are completely avoided(i.e.,chloroform,isopropanol).In addition,the extracted fungus DNA has good integrity and high quality,which meet regular PCR amplification for gene cloning or transformant identification.This method exhibits promising prospects for the future utilization.