Mapping ESTs by Fiber–FISH

A visual transcript map of six genes was constructed on the chromosome 21q22.3 by high resolution fluorescence in situ hybridization (FISH). Expressed sequence tags (ESTs) from six genes— PWP2 , KNP1 , AIRE , C21orf3 , SMT3A , and C21orf1 —were successfully localized by fiber–FISH by use of sensitive tyramide-based detection. The sizes of the ESTs varied between 315 to 956 bp and most of them map within the 3′-untranslated region. The ESTs were assigned to and subsequently ordered within cosmid, PAC, and BAC clones hybridized on DNA fibers. Physical distances between ESTs and known markers were determined. Our results demonstrate the feasibility and accuracy of visual mapping EST sequences in relation to known markers. The main advantage of this approach is that it can be applied to finely map any of the database ESTs for positional cloning efforts. The sensitivity, specificity, and reproducibility of this high-resolution EST mapping technique is evaluated.