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. characterized in rat a decade ago [2] and have since been detected in many vertebrate species [reviewed in 6]. So far, about 3000 OR genes and pseudogenes are known in 24 vertebrate species [7][8][9][10][11][12]. They are divided into 32 distinct families based on phylogenetic analysis [8].Roughly 900 OR coding sequences were found in the first draft of the human genome [9,10]. As predicted [7,13], between 53% and 63% of them have frame disruptions and are therefore considered as pseudogenes. OR genes are found on almost all human chromosomes except chromosome 20 and Y [7,9,10,13,14]. Almost 80% of the ORs are organized in clusters of six or more genes [9,10]. This is in good agreement with previous fluorescence in situ hybridization (FISH) experiments and sequencing data [7,13,14]. 296Article doi:10.1006/geno.2002.6830, available online at http://www.idealibrary.com on IDEAL The coding region of OR genes spans approximately 1 kb. This region lacks introns [2,13] and is preceded by a large intron and several short noncoding exons [16][17][18][19][20]. Inside the coding region there are several conserved segments [21] that allow easy amplification of the intronless coding region from genomic DNA by PCR assay. The PCR product is termed the "olfactory receptor sequence tag" (OST) [7].We developed and experimentally tested a practical scheme for deciphering families of genes (DEFOG). The scheme provides a powerful means to obtain a sequence composition of a gene family in species for which high-throughput genomic sequencing data are unavailable. To validate DEFOG, we tested it on the human OR gene superfamily. Starting with a limited number of human ORs, it almost tripled the size of this set in a single experiment. We suggest that DEFOG can be successfully appli...

Faster pattern matching with character classes using prime number encoding

“…Degenerate primers can be used to amplify several related genomic sequences in a single PCR experiment. We studied the computational problem of designing highly degenerate primers [15], and applied our algorithms in experiments for studying the human and canine olfactory receptor genes [16,17]. A common problem in the design of degenerate primers is to verify that the primers do not bind to DNA regions others than those they are meant to amplify.…”

Comparison of the fraction of olfactory receptor pseudogenes in wolf (Canis lupus) with domestic dog (Canis familiaris)

“…A set of degenerate primers were designed on the basis of the H2 (forward primer) and H7 (reverse primer) domains of all human OR genes according to the HORDE database, using the program DEFOG (Fuchs et al 2002;Matasci 2003). The primer set PC1 was designed on all Class I Ors, while the primer set PC2 was designed on all Class II OR genes.…”

MEPD: a resource for medaka gene expression patterns

“…OFP is insensitive to the presence of related repeats in transcripts from unrelated genes, because fingerprints are calculated on the basis of entire clone inserts. Also, clustering parameters can be adjusted to distinguish members of multigene families (Fuchs et al, 2002). In MEPD, we have 18 364 sequence entries of this library, 10 461 of which represent the unigene set.…”