Entwicklung analytisch-molekularbiologischer Verfahren zur Konstruktion einer Plasmid-Genbank aus Boden-DNA in Escherichia coli und deren Durchmusterung nach neuen Enzymen für die technische Anwendung [article]

Hubert Zipper, Universität Stuttgart, Universität Stuttgart
2005
The immense reservoir of untapped microbial diversity is regarded as a source for new enzymes. Traditional approaches to isolate novel enzymes by cultivation of microorganisms from soil samples revealed to be incomplete as only a minor proportion of microorganisms are culturable under standard laboratory conditions. Thus, the access to genomes of microbial communities via gene banks is an important key for the identification of new enzymes. In the present work, the principle approach for the
more » ... approach for the generation of genomic preparations from microbial communities involved the recovery of DNA from soil samples, the quantification of DNA, the purification of the soil-DNA, and its subsequent cloning into E. coli in order to screen the gene bank for hydrolases and oxidoreductases useful for technical application. DNA extracts from sediment samples are often contaminated by humic acids which interfere with many molecular biological applications, e.g. DNA quantification, enzymatic DNA modification. In order to develop a novel method for DNA quantification in crude extracts, the interaction between DNA and the fluorescent dye SYBR Green I (SG) was studied first. The analysis of the structure of SG (2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium) and of the molar absorption coefficient (about 73000 (M·cm)-1 at 494 nm) enabled interaction studies with DNA which revealed the occurrence of (semi-)intercalation, followed by surface binding at dye/base pair ratios above 0,15. Only the latter binding led to a significant increase in fluorescence. Studies with poly(dA)poly(dT) and poly(dG)poly(dC) homopolymers indicated minor groove binding. On the basis of these results mechanisms underlying the impact of humic acids on DNA quantification by SG and consequences for the analysis of soils and aquatic sediments were investigated. According to these studies the effect of humic acids was based on an inner filter effect, collisional quenching, and competitive bindin [...]
doi:10.18419/opus-1657 fatcat:ruk7jhodnracnl4ibqwgr5mg4a