Identification of genes using oligonucleotides corresponding to splice site consensus sequences.
The identification of genes in genomic DNA presents challenging technical difficulties. We show here the feasibility of using short oligonucleotides based on the consensus sequences surrounding intron-exon junctions to detect random phage and cosmid clones containing genes both through the analysis of DNA blots and by direct screening. Three degenerate oligonucleotides, a 10-mer corresponding to the 5' splice junction and a 9-mer and a 15-mer corresponding to the 3' splice junction, were tested on the known intron-exon boundaries of the cloned human proteolipid protein (PLP) gene at hybridization and washing temperatures appropriate to their length and composition. All predicted hybridizations were observed. The oligonucleotides were also used to identify random genomic plasmid and cosmid clones containing putative intron-exon junctions; the presence of genes in these clones was supported by RNA blot analysis and by cross-hybridization to DNA from other species. This technique should facilitate the identification of genes for inherited diseases by positional cloning studies and will assist in the identification of genes in random clones for the human genome project.
Melmer
G
G
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Buchwald
M
M
D001483:Base Sequence; D002460:Cell Line; D002897:Chromosomes, Human, Pair 7; D004247:DNA / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000235:genetics*; D018991:Myelin Proteolipid Protein; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012326:RNA Splicing; D012333:RNA, Messenger
D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA