Direct selection: a method for the isolation of cDNAs encoded by large genomic regions.
We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles. This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene. Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment. More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched. We confirmed that this cDNA was encoded by the yeast artificial chromosome. Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library. DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins. Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene. The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome.
Lovett
M
M
Department of Molecular Genetics, Genelabs Inc., Redwood City, CA 94063.
Kere
J
J
Hinton
L M
LM
D001483:Base Sequence; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004921:Erythropoietin / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics*; D015723:Gene Library; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping
D009841:Oligonucleotides; D004921:Erythropoietin; D004247:DNA; D019204:GTP-Binding Proteins