Isolation of CpG islands from large genomic clones.
Positional cloning is a powerful method for the identification of genes. Using genetic and physical mapping methods the genomic region within which a particular gene is located can relatively easily be narrowed down to a comparatively small area contained within cosmid, PAC or BAC clones. It is then a matter of identifying genes within these clones. Here we describe the appli-cation of a technique, which has been successfully used for the bulk purification of CpG islands from whole genomes, to the isolation of CpG island sequences from such clones. As CpG islands overlap transcription units they can be used to isolate full-length cDNAs for associated genes, either by probing cDNA libraries or by searching databases. CpG islands are linked with approximately 60% of human genes and because their isolation is independent of the expression profile of these genes this approach would complement other expression-based methods of gene identification. By applying this technique to a cosmid clone known to contain the PAX6 gene we successfully isolated the CpG island for this gene along with other CpG island-like sequences. Closer examination revealed that an extensive genomic region around the 5'-end of PAX6 is unusual with regard to methylation and GC content. CpG island sequences were also successfully isolated from a PAC clone carrying the MBD1 gene. These included the complete CpG island containing the first exon and regulatory sequences from MBD1.
Cross
S H
SH
Institute of Cell and Molecular Biology, University of Edinburgh, Darwin Building, King's Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK. sally.cross@hgu.mrc.ac.uk
Clark
V H
VH
Bird
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AP
D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D018899:CpG Islands; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005136:Eye Proteins; D008040:Genetic Linkage; D015894:Genome, Human; D018398:Homeodomain Proteins; D006801:Humans; D008969:Molecular Sequence Data; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D015183:Restriction Mapping
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins