Use of interspersed repetitive sequences-PCR products for cDNA selection.
In order to increase the efficiency of cDNA selection approaches, we describe the use of interspersed repetitive sequences-PCR (IRS-PCR) products to isolate genes from large-insert genomic clones. IRS-PCR is conducted on total yeast DNA containing a YAC of interest so that there is no need to purify the starting genomic clone. This enables the production of large amounts of genomic substrate for cDNA selection and allows the use of unstable YAC clones. Moreover, the hybridization of the IRS-PCR product to the cDNA clones after selection introduces a positive selection step. We tested these PCR products from YACs for the presence of exons, using cDNAs originating from seven different genes. In each case, at least one exon was present in the IRS-PCR product. We have applied this strategy to four YAC clones originating from the human X Chromosome (Chr). All the selected cDNAs, strongly positive with the IRS-PCR product, did indeed originate from a gene in the region covered by the YAC. In all cases, the previously known genes contained in the genomic clones have been isolated. In addition, we have isolated human genes that have already been described but not assigned to any chromosomal region.
Villard
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INSERM U406. 27, Marseille, France.
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D001483:Base Sequence; D002874:Chromosome Mapping; D018244:Chromosomes, Artificial, Yeast; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005091:Exons; D006801:Humans; D008969:Molecular Sequence Data; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000235:genetics; D050810:Octamer Transcription Factors; D016133:Polymerase Chain Reaction / Q000379:methods*; D016601:RNA-Binding Proteins / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D014960:X Chromosome
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D050810:Octamer Transcription Factors; D016601:RNA-Binding Proteins