Improved simple generation of GTG-band specific painting probes.

We developed an improved, simple method of generating chromosome-region-specific probes from only a few microdissected chromosomes. One to five dissected fragments from a defined chromosomal region were processed with a PEG/proteinase K cycling deproteinization step and directly amplified with a two-step amplification system using a degenerate oligonucleotide primed shuttle polymerase chain reaction (DOP-Shuttle-PCR). This modified method offered three advantages over previously reported methods: relaxation of the highly condensed chromosomal DNA, reduction of the risk of endogenous and exogenous contamination, and high efficiency amplification of template DNA. High intensity in the fluorescence in situ hybridization (FISH) signals from normal metaphase chromosomes, as well as regional specificity of these probes, corresponding to regions on R-banded chromosomes, were observed.