title: DEFOG: a practical scheme for deciphering families of genes.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and the Crown Human Genome Center, The Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Tania
authors.initials: T
authors.lastname: Fuchs
authors.forename: Barbora
authors.initials: B
authors.lastname: Malecova
authors.forename: Chaim
authors.initials: C
authors.lastname: Linhart
authors.forename: Roded
authors.initials: R
authors.lastname: Sharan
authors.forename: Miriam
authors.initials: M
authors.lastname: Khen
authors.forename: Ralf
authors.initials: R
authors.lastname: Herwig
authors.forename: Dmitry
authors.initials: D
authors.lastname: Shmulevich
authors.forename: Rani
authors.initials: R
authors.lastname: Elkon
authors.forename: Matthias
authors.initials: M
authors.lastname: Steinfath
authors.forename: John K
authors.initials: JK
authors.lastname: O'Brien
authors.forename: Uwe
authors.initials: U
authors.lastname: Radelof
authors.forename: Hans
authors.initials: H
authors.lastname: Lehrach
authors.forename: Doron
authors.initials: D
authors.lastname: Lancet
authors.forename: Ron
authors.initials: R
authors.lastname: Shamir
mesh_terms: D000465:Algorithms; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D005810:Multigene Family; D018035:Receptors, Odorant / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D018035:Receptors, Odorant