A new FISH protocol with increased sensitivity for physical mapping with short probes in plants

Flavia Guzzo, Evelyn Campagnari, Marisa Levi
2000 Journal of Experimental Botany  
nuclei, and to map DNA sequences on chromosomes. Mapping resolution is higher on pachytene chromosomes Fluorescence in situ hybridization (FISH) is a wellthan on metaphase chromosomes (Shen et al., 1987; established technique used for the detection of Zhong et al., 1996), due to their less contracted structure; specific DNA regions, that has been applied to it is even higher in interphase nuclei (Lawrence et al., interphase nuclei, pachytene and metaphase chromo-1988, 1990 Trask et al., 1989
more » ... sk et al., , 1991. In the latter case, somes as well as to extended DNA fibres. This techhowever, the possibility of identifying individual chromonique allows the physical mapping of specific DNA somes is lost. The use of FISH on extended DNA fibres sequences both on individual chromosomes and further enhances the physical mapping resolution (de extended fibres. A new FISH protocol is described here Jong et al., 1999), due to the high linear stretching degrees that enhances the sensitivity of the method. Probes of chromatin, evaluated to be around 3.27 kb mm−1, for small unique DNA sequences of less than 2 kb give according to Fransz et al. (Fransz et al., 1996a). high signal-to-noise ratio with this method, and can The sensitivity of the FISH techniques so far developed be visualized easily by means of conventional fluoreshas allowed the identification of single DNA sequences cence microscopy. as small as 0.25 kb on human chromosomes (Richards et al., 1994). However, FISH on plants with small probes Key words: FISH, Asparagus officinalis, physical mapping. ( less than 10 kb) seems to be more difficult to perform. The reports on FISH with probes for small single copy DNA targets (1-2 kb) are limited in number. The best
doi:10.1093/jexbot/51.346.965 pmid:10948224 fatcat:ti4un7k4g5dglh2wvl7x3j7miy