【摘要】 建立了一种土壤DNA提取方法。根据DNA产量和纯度2个评价指标,从3种手提土壤DNA方法中优选出方法B为手提DNA方法(Labmethod),它包括样品预处理,细胞裂解,粗DNA纯化,其中细胞裂解组合了玻璃珠击打,SDS裂解,溶菌酶裂解。进一步应用PCR-限制性片段长度多态性(Restrictionfragmentlengthpolymorphism,RFLP)技术及PCR-温度梯度凝胶电泳(Temperaturegradientgelelectrophoresis,TGGE)技术,结合DNA产量、纯度、片段大小以及所反映的微生物群落结构特性等指标评价了手提方法(Labmethod)得到的总DNA质量,并将这些结果与2种应用较广的商业试剂盒(MoBioUltraCleanSoilDNAKit和Bio101FastDNASPINKit(ForSoil))所得DNA的各种指标进行了比较。结果表明,手提方法(Labmethod)的粗DNA产量低于Bio101Kit的,但高于MoBioKit的。这些方法所得DNA的长度都在21kb左右,Labmethod提取的DNA没有严重被剪切现象,而2种试剂盒提取的DNA都有不同程度的剪切。手提方法得到的DNA经纯化后,应用细菌及真菌特异引物进行PCR扩增,均能获得目的片段,表明该方法能从土壤中同时有效提取细菌和真菌总基因组DNA。并且,手提方法所得DNA的细菌16SrDNA和真菌18SrDNA的PCR-RFLP图谱与两种商业试剂盒的图谱基本相似,但3种方法提取的DNA在细菌16SrDNAV3区和真菌28SrDNA片段的PCR-TGGE图谱上都存在一定差异,主要表现在一些弱势条带的有无或强弱上。这说明手提方法提取的总DNA与2种商业试剂盒一样,在一定程度上能反映微生物群落的多样性和组成。总之,手提DNA方法(Labmethod)所用试剂普通,价格便宜,能在4h以内获得够质够量的土壤DNA用于微生物分子生态学研究,较适合于广大普通实验室进行土壤DNA提取工作。更多还原

【Abstract】 A method of extracting soil total DNA was developed. Using DNA fingerprinting methods could detect diverse members of soil microbial consortia, including those microorganisms not yet cultivated. However, the extraction of DNA from soil is not simple. Soil DNA extraction method B was selected from three manual DNA extraction methods as Lab Method, based on DNA yield and purity. The Lab Method comprised sample pre-treatment, cell lyses and crude DNA purification. Cell lyses involved in bead beating, SDS and lysozyme treatment. The Lab Method was valuated by comparing with Mo Bio UltraClean Soil DNA Kit and Bio 101 FastDNA SPIN Kit (For Soil) in DNA yield, purity, fragment size, and characterize of microbial community structure reflected by PCR-Restriction fragment length polymorphism (PCR-RFLP) and PCR-Temperature gradient gel electrophoresis (PCR-TGGE) techniques. The crude DNA yield using Lab Method was lower than that of Bio 101 Kit, but higher than that of Mo Bio Kit. Most of the isolated DNA by the three methods was near 21-kilo bases in size, and the lab method led to less cleavage in DNA fragment. After purified, the total DNA extracted by Lab Method was competent for PCR amplification with bacterial and fungal specific primers, demonstrating Lab Method was suit for simultaneous recovery of bacterial and fungal community total DNA from agricultural soils for studying microbial community structure, function and dynamics. Bacterial 16S rDNA and fungal 18S rDNA PCR-RFLP profiles of DNA samples isolated using the three methods were similar, while there were some differences in the PCR-TGGE profiles of bacterial 16S rDNA V3 and fungal 28S rDNA regions among the three kinds of DNA samples, mainly displayed in the intensity and absent of some faint bands. It was suggested that the composition of retrieved total DNA by Lab Method could reflect the microbial community in soil to some extent similar as by using two kits, so Lab Method was feasible for most labs for soil DNA extraction for its low cost and not time-consuming.更多还原