ausblenden:
Schlagwörter:
-
Zusammenfassung:
We developed a novel efficient scheme, DEFOG (for "deciphering families of genes"), for determining sequences of numerous genes from a family of interest. The scheme provides a powerful means to obtain a gene family composition in species for which high-throughput genomic sequencing data are not available. DEFOG uses two key procedures. The first is a novel algorithm for designing highly degenerate primers based on a set of known genes from the family of interest. These primers are used in PCR reactions to amplify the members of the gene family. The second combines oligofingerprinting of the cloned PCR products with clustering of the clones based on their fingerprints. By selecting members from each cluster, a low-redundancy clone subset is chosen for sequencing. We applied the scheme to the human olfactory receptor (OR) genes. OR genes constitute the largest gene superfamily in the human genome, as well as in the genomes of other vertebrate species. DEFOG almost tripled the size of the initial repertoire of human ORs in a single experiment, and only 7% of the PCR clones had to be sequenced. Extremely high degeneracies, reaching over a billion combinations of distinct PCR primer pairs, proved to be very effective and yielded only 0.4% nonspecific products.
