不同提取缓冲液对红树林土壤DNA提取品质的影响

摘要

针对1种典型的酸性、高有机质含量类型土壤-红树林土壤,从DNA产量、DNA纯度、土壤腐殖酸类物质的提取量以及DNA提取物PCR(polymerase chain reaction)扩增反应的敏感度等方面比较了不同提取缓冲液对土壤DNA品质的影响.结果表明,酸性SDS(sodium dodecyl sulfate)提取缓冲液所获得的土壤DNA产量及纯度均较低;PVP (polyvinylpyrrolidone)提取缓冲液所获得的土壤DNA产量较高,但土壤腐殖酸类物质的提取量亦远高于其他提取缓冲液;酸性SDS提取缓冲液及PVP提取缓冲液所获得的土壤DNA提取物即使稀释到10-3量级亦不能获得目的基因PCR扩增条带.PVPP(polyvinylpolypyrrolidone)提取缓冲液及CTAB(cetyltrimethylammoniumbromide)+PVPP提取缓冲液所获得的土壤DNA产量较PVP提取缓冲液低,但其纯度较高,并且其纯度随提取缓冲液中PVPP浓度的升高而提高;当2%PVPP提取缓冲液及CTAB+PVPP提取缓冲液所获得的土壤DNA提取物分别稀释到10-2及10-1量级时,均能获得PCR扩增条带.综上所述,酸性提取缓冲液及PVP提取缓冲液不适用于酸性、高有机质含量类型土壤DNA的提取,而2%PVPP提取缓冲液及CTAB+PVPP提取缓冲液均能提高此类土壤DNA的提取品质.%The authors compared the influence of six different extraction buffers on DNA yields, DNA purity, co-extracted humic compound yields, and their PCR (polymerase chain reaction) sensitivity from a typical acidic and high organic content soil type, mangrove soil.Results showed that the quality of the soil DNA obtained using the acidic SDS (sodium dodecyl sulfate) buffer was poor.The DNA yields obtained using the PVP (polyvinylpyrrolidone) buffer were higher than the other buffers, whereas the co-extracted humic compound yields were significantly higher.PCR results showed that no expected bands were obtained even in 1:1000 dilutions of the DNA extracts obtained using the acidic SDS buffer or PVP buffer.On the other hand, although the PVPP (polyvinylpolypyrrolidone) buffer and the CTAB (cetyltrimethylammonium bromide) and PVPP buffer resulted in a lower DNA yield, the purity of the soil DNA was improved.In addition, the purity was improved as the concentration of PVPP in the buffer increased.Moreover, PCR amplification was successfully conducted in the 1:10 dilutions of the DNA extracts obtained using the CTAB and PVPP buffer and in the 1:100 dilutions of DNA extracts obtained using the 2％ PVPP buffer, respectively.In conclusion, acidic SDS buffer and PVP buffer were not suitable for extracting DNA from the acidic and high organic content soil type; however, 2％ PVPP buffer and CTAB and PVPP buffer improved the DNA quality.