PCR-mediated cloning of HpaII tiny fragments from microdissected human chromosomes

Abstract

Vertebrate DNA contains a small fraction of unmethylated CpG-rich DNA sequences, many of which include the 5' end of a gene. This fraction can be detected by its cleavage to tiny fragments with the methylation-sensitive restriction enzyme HpaII. Thus, the isolation of HpaII tiny fragments (HTFs) from a specific chromosome region may be a useful approach for making an inventory of the genes contained in it. Using microdissection, we have isolated DNA from human chromosome band 8q24.1. The DNA was digested with HpaII, ligated to a ClaI-cut pUC plasmid, and amplified with Taq DNA polymerase and the standard M13/pUC forward and reverse sequencing primers. The amplification products were used to construct an HTF library, which is enriched for CpG-rich single-copy clones.