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21. Detection and Isolation of Selected Genes of Interest from Metagenomic Libraries by a DNA Microarray Approach
A DNA microarray-based approach is described for screening metagenomic libraries for the presence of selected genes. The protocol is exemplified for the identification of flavin-binding, blue-light-sensitive biological photoreceptors (BL), based on a homology search in already sequenced, annotated genomes. The microarray carried 149 different 54-mer oligonucleotides, derived from consensus sequences of BL photoreceptors. The array could readily identify targets carrying 4% sequence mismatch, and allowed unambiguous identification of a positive cosmid clone of as little as 10 ng against a background of 25 μg of cosmid DNA. The protocol allows screening up to 1,200 library clones in concentrations as low as ca. 20 ng, each with a ca. 40 kb insert size readily in a single batch. Calibration and control conditions are outlined. This protocol, when applied to the thermophilic fraction of a soil sample, yielded the identification and functional characterization of a novel, BL-encoding gene that showed a 58% similarity to a known, BL-encoding gene from Kineococcus radiotolerans SRS30216 (similarity values refer to the respective LOV domains).
Affiliation(s): (1) Max Planck Institute for Bioinorganic Chemistry, Mülheim, Germany
Series: Methods in Molecular Biology  |  Volume: 668  |  Pub. Date: Sep-15-2010  |  Page Range: 299-312  |  DOI: 10.1007/978-1-60761-823-2_21
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