[Extraction of DNA from environmental samples and construction of mixed genomic DNA libraries].
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 16 micrograms per gram of dry sample. Mixed genomic DNA libraries for two of the environmental samples were constructed by inserting restriction fragments (3-8 kb) of the purified DNAs into plasmid pUC18 and transforming E. coli DH5 alpha with the resultant plasmids. Approximately 10(3) to 10(4) insert-containing clones were obtained from 1 g of each 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.
Wang
X
X
Department of Biochemistry & Molecular Biology, College of Life Sciences, Beijing University, Beijing 1000871, China.
Tang
Y
Y
Wang
J
J
Huang
Y
Y
Chen
R
R
Huang
L
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D015698:Genomic Library; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D013880:Thioredoxin-Disulfide Reductase