A new approach to high resolution FISH

Various gene mapping efforts need high resolution ordering of clones. Using fluorescence in situ hybridization (FISH) best resolution has so far been achieved by the methods where the cells have been lysed and the chromatin has been stretched on the slide. This original DIRVISH method can resolve probes located only a few kilobases apart. We describe here another, novel alternative for high resolution FISH mapping. In this method agarose embedded DNA in form of a pulse field gel block is used as a hybridization target. This target is easy to prepare and available in many laboratories dealing with positional cloning. A small piece of PFGE block is melted on a microscope slide, the DNA is stretched and air-dried. In addition to the technical simplicity of the method it also offers possibilities for pretreatments of the target DNA such as cleavage by rare cutting restriction enzymes. The modification has significantly improved the reproducibility, hybridization efficiency and quality of hybridization signals in the stretched DNA. In practice this method has been applied in positional cloning of genes behind two variants of neuronal lipofuscinosis (INCL, Chr 1p and vINCL, Chr 13q). Here we have successfully ordered YACs, cosmid, phage and plasmid clones onmore » genomic DNA facilitating rapid construction of physical maps of the two areas. DNA released from YAC-blocks has also been used as target for ordering cosmid and phage clones within the YAC.« less