环境样品中DNA的分离纯化和文库构建

EXTRACTION OF DNA FROM ENVIRONMENTAL SAMPLES AND CONSTRUCTION OF MIXED GENOMIC DNA LIBRARIES

采用研磨 /冻融和SDS/蛋白酶K热处理等理化方法 ,直接从性质不同的环境样品中提取和纯化混合基因组DNA。所获得纯品DNA的产量为每克样品 2～ 1 6μg。对纯品DNA进行限制性内切酶处理后 ,构建了以pUC1 8为载体的DNA文库。建库效率为从每克环境样品获得约 1 0 3～ 1 0 4 个含 3～ 8kb外源随机插入片段的克隆。通过DNA序列测定和基因注释 ,对从文库中随机选取的克隆进行了分析 ,发现外源插入片段均含序列未见报道的新基因。本文所做的尝试对于保存。

A method has been developed for extracting and purifying genomic DNA from environmental samples.In this method,an environmental sample is treated first by grinding and freezing/thawing and subsequently by SDS/proteinase K\|based DNA extraction.The yields of purified DNA from three samples used in this study ranged from 2 to 16μg per gram of dry sample.Mixed genomic DNA libraries for two of the environmental samples were constructed by inserting restriction fragements (3～8 kb) of the purified DNAs into plasmid pUC18 and transforming \%E.coli\% DH5α with the resultant plasmids.Approximately 10 3 to 10 4 insert\|containing clones were obtained from 1g of cach sample.Clone libraries were analyzed by DNA sequencing and gene annotation.Among 20 randomly\|selected clones, 14 contained an insert whose sequence had not been reported while the rest had an insert of either E.coli or vector origin.A search of sequence databases using the end sequences of each of the foreign inserts showed that each sequence was part of a gene encoding,in most cases, a predictable function.Our results are of significance to the collection,investigation and exploitation of the genes of uncultured microorganisms.