Microscopy and image analysis of fibre-FISH

Abstract

In this paper the aspects of image acquisition, processing and analysis for DNA-fibre mapping are described. As the nature and the quality of the fibre-FISH signals (given its resolution range of 1–500 kb) may vary to a great extent, an interactive approach was chosen for the selection and analysis of the fibres. The accuracy of this fibre-FISH mapping approach was compared with restriction mapping on the basis of a map of seven cosmid contigs from the thyroglobulin gene, which spans about 300 kb. The results were in full agreement with restriction mapping. Standard errors for sizes of the cosmids, gaps, and overlaps were obtained between 2.0 and 6.2 kb. By alternately labelling the clones of the DNA map a colour barcode can be composed which eases the identification of gene rearrangements, as is illustrated on two patients with a deletion in the Duchenne muscular dystrophy (DMD) gene. The time needed for straightening a fibre and defining the distances between the different cosmids is dominated by the amount of human interaction and typically takes 1–2 min. From this study it is clear that fibre-FISH analysis is well suited for mapping cosmid contigs and defining breakpoints in patient material with the same or better accuracy as restriction mapping and PCR analysis.