SUBTRACTIVE HYBRIDIZATION CLONING FOR PROBES FOR GENE ISOLATION AND MAPPING

摘要

Simplified methods to isolate eukaryotic genes based on their genomic location are provided. The methods utilize probes that are enriched in a sequence of a target eukaryotic gene. The probes are obtained by subtractive hybridization utilizing at least two hybrid cell lines which are nearly isogenic. The hybrid cell lines contain genetic sequences from a eukaryotic chromosome that is heterologous to the host cell, but differ primarily in the sequence of the target gene within the heterologous eukaryotic chromosome. Using the lines, probes that are enriched for sequences encoded in the region of non-overlap between the provided somatic cell hybrid lines are generated by subtractive hybridization. The subtractive hybridization of the method does not rely only on differential expression. Rather, the 3'-sequence divergence between the transcripts of the non-overlap region of the heterologous chromosome and the transcripts of the hybrid host recipient cell allows the specific removal of host sequences from the probe population. Thus, the method overcomes the problem of homology of genes between the targeted gene and the analogous gene (if any) in the recipient hybrid host cell.