Detection of small, single-copy genes on protein-G-banded chromosomes by electron microscopy

摘要

A method for the detection by electron microscopy of chromosome banding after in situ hybridization of small, nonradioactive DNA sequences is described. Typical high-resolution G-banding is produced by adding 5-bromodeoxyuridine (BrdU) during the last part of the S-phase and by applying a monoclonal antibody against the BrdU-substituted chromosome segments, followed by the addition of protein G, but no further treatment. A protocol for in situ hybridization of small, single-copy biotinylated DNA sequences and their detection by immunogold tagging on banded chromosomes is also described. This combined approach permits high-resolution mapping of small DNA sequences and should be useful in discriminating between neighboring DNA fragments.