FISH of Alu-PCR-Amplified YAC Clones and Applications in Tumor Cytogenetics

Yeast artificial chromosomes (YACs) (1 ) containing human inserts of up to 1 megabase (Mb) length have been mapped by fluorescence in situ hybridization (FISH) (for review see ref. 2 ). If total yeast clone DNA is used as a probe, an excess of yeast DNA (approx 98%) is labeled in addition to the human sequences (approx 2%). This excess labeling not only leads to wastage of expensive labeling reagents, but contributes to background in in situ hybridization experiments. The hybridization efficiency is often less than satisfactory in metaphase spreads, whereas signals in interphase nuclei generally cannot be evaluated. Cutting out the YAC band from a pulsed-field gel and purification of the DNA is time-consuming and yields only rather limited amounts of a probe.