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).
Pathak
Gopal P
GP
Max Planck Institute for Bioinorganic Chemistry, Mülheim, Germany.
Gärtner
Wolfgang
W
D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D005798:Genes, Bacterial; D054892:Metagenome; D056186:Metagenomics / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D016415:Sequence Alignment
D004269:DNA, Bacterial