[Rapid isolation of CpG-islands by PCR-amplification of genomic DNA fragments using a "CpG-enriched" primer]

One of the main problems of positional cloning of human disease genes is isolation and identification of coding DNA segments in large genomic regions. Traditional methods of isolation of cDNA fragments usually do not allow detection of transcribed sequences including 5' ends of genes. In this study, a method of rapid isolation of CpG islands associated with 5' regulatory segments of the majority of mammalian genes is proposed. This method, which is termed the CGE-PCR method, is based on the use of a CpG-enriched oligonucleotide primer and a "buble" or Alu primer in PCR amplification of human genomic DNA fragments cloned in P1 clones or cosmids. The method was applied to cloning CpG-enriched sequences mapped in the region in which the gene for the early-onset familial form of Alzheimer's disease type III is located (14q24.3). The majority of cloned PCR products contained CpG islands. Detection of clone localization in chromosomal region 14q24.3 was performed by YAC mapping. In total, nine CpG-enriched nucleotide sequences that are potential 5' regions of different genes were detected and mapped by the CGE-PRC-.