Isolation of CpG islands from large genomic clones

Positional cloning is a powerful method for the identification of genes. Us ing genetic and physical mapping methods the genomic region within which a particular gene is located can relatively easily be narrowed down to a comp aratively small area contained within cosmid, PAC or BAC clones. It is then a matter of identifying genes within these clones. Here we describe the ap plication of a technique, which has been successfully used for the bulk pur ification of CpG islands from whole genomes, to the isolation of CpG island sequences from such clones. As CpG islands overlap transcription units the y can be used to isolate full-length cDNAs for associated genes, either by probing cDNA libraries or by searching databases. CpG islands are linked wi th similar to 60% of human genes and because their isolation is independent of the expression profile of these genes this approach would complement ot her expression-based methods of gene identification, By applying this techn ique to a cosmid clone known to contain the PAX6 gene we successfully isola ted 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 isl and sequences were also successfully isolated from a PAC clone carrying the MBD1 gene. These included the complete CPG island containing the first exo n and regulatory sequences from MBD1.