深海沉积物中微量DNA的提取及应用

SMALL-SCALE DNA EXTRACTION FROM DEEP SEA SEDIMENT AND APPLICATION

采用化学裂解和酶解相结合的方法 ,进行了深海沉积物中微量DNA的提取 ,并采用DNA吸附树脂进行纯化。结果表明 ,该方法能有效地去除沉积物中的腐殖酸等抑制剂 ,每克湿重沉积物样品可得到DNA约 1 6 μg,回收率可达 95 % ,所得到的DNA分子片段均在 2 3kb左右 ,纯化后的DNA可直接应用于各种分子生物学操作。利用细菌 1 6SrDNA通用引物对所提取的深海沉积物DNA进行了PCR RFLP及系统发育分析 ,各主要细菌类群均能检出 ,证实该方法可以应用于深海等极端环境中微生物的多样性调查、系统发育分析以及特殊功能基因的筛选 。

In recent years, several protocols based on the extraction of DNA from environmental samples have been developed for the microbial diversity study in nature. We report an efficient and nonselective protocol to extract the small-scale DNA from deep sea sediment with minimal shearing. The direct lysis technique involving SDS and lysozyme treatments was used because it could yield more DNA with less bias from sediment. The addition of PVPP (polyvinylpolypyrrolidone) and CTAB (Cetyl-trimethyl-ammonium Bromide) could remove the humic acid and other inhibitors from sediment efficiently. About 16 microgramme pure DNA with the size of 23kb, which could be used directly for molecular operation, was recovered from one gram deep sea sediment after purification by resin. The DNA recovery ratio was about 95%. PCR-RFLP (Restriction Fragment Length Polymorphism) analysis was done by universal bacterial 16S rDNA primers using the purified total DNA as template. The analysis of 16S rDNA sequences indicated that the majority of bacteria taxa in the sample could be detected. The method could be applied in analyzing the diversity and phylogenesis of natural microbial communities in extreme environment with low biomass; in screening for gene with special function; and in semi-quantifying the biomass of environmental sample.