一种直接用于PCR分析的风沙土微生物总DNA提取方法

以风沙土为材料,直接提取土壤微生物DNA.采用SDS—溶菌酶—CTAB—蛋白酶K和液氮反复冻融法裂解细胞得到DNA粗提液,以PEG对其纯化.结果表明,SDS—溶菌酶—CTAB—蛋白酶K和液氮反复冻融法裂解细胞可获得大片段的DNA,提高DNA产率.每克干土的DNA提取量为0.586～1.311μg.所提DNA片段在23Kb以上.PEG可有助于去除DNA中腐植酸,DNA不作回收纯化即可作为模板进行16SrDNAPCR扩增.SDS—溶菌酶—CTAB—蛋白酶K和液氮反复冻融裂解细胞、PEG纯化DNA的方法组合是一种简便的适合风沙土微生物总DNA的提取方法.

A Method for Total Microbial DNA Extraction Directly Meeting the Requirement of PCR Amplification from an Aeolian Sandy Soil

The soil microbial DNA was directly extracted from aeolian sandy soil. The DNA crude extracts were obtained by the cell lysis via the repeated freezing-thawing process of SDS, lysozyme, CTAB, proteinase K and liquid nitrogen in combination with the crude extracts purified by PEG (polyethylene glycol). In this way the large fragment DNA was obtained from the lytic cells with the DNA yield increased, i.e., 0.586~1.311 μg · g^-1(dry soil). In addition, the fragment size of DNA was over 23 Kb, and PEG was helpful to remove humic acid in DNA effectively. The unpurified DNA could be directly used as templates for PCR amplification of 16S rDNA. The method mentioned above was simple and appropriate for DNA extraction from the aeolian sandy soil.