The study of preparing probes for Fluorescence in situ Hybridization (FISH) from YAC clones by universal primer PCR

A simple and efficient method, universal primer PCR (UP PCR) is described, that permits preparing DNA probes for FISH from a trace amount of YAC DNA for which sequence does not need to be known. That is, Linking the annealed UP-Linker to the YAC DNA digested with Alu I after isolation by pulse field gel electrophresis (PFGE), then amplifying and labelling the YAC DNA by PCR using universal primer (UP). Because the restriction sites of Alu I are very frequent in the human genome and UP (25nt) is much longer than Linker (11nt), the probes generated by UP PCR can cover the full length of inserted DNA and PCR amplification itself can achieve changes from extensiveness to specificity. FISH on lymphocytes using human chromosome 21 probes prepared by UP PCR gives bright hybridization signals and correct numbers of chromosome 21 in more than 98.4% of interphase nuclei and 99.6% of metaphases, respectively.