Chromosome Painting by GISH and Multicolor FISH
Fluorescent in situ hybridization (FISH) is a powerful cytogenetic technique for identifying chromosomes and mapping specific genes and DNA sequences on individual chromosomes. Genomic in situ hybridization (GISH) and multicolor FISH (mc-FISH) represent two special types of FISH techniques. Both GISH and mc-FISH experiments have general steps and features of FISH, including chromosome preparation, probe labeling, blocking DNA preparation, target-probe DNA hybridization, post-hybridization washes, and hybridization signal detection. Specifically, GISH uses total genomic DNA from two species as probe and blocking DNA, respectively, and it can differentiate chromosomes from different genomes. The mc-FISH takes advantage of simultaneous hybridization of several DNA probes labeled by different fluorochromes to different targets on the same chromosome sample. Hybridization signals from different probes are detected using different fluorescence filter sets. Multicolor FISH can provide more structural details for target chromosomes than single-color FISH. In this chapter, we present the general experimental procedures for these two techniques with specific details in the critical steps we have modified in our laboratories.
Key wordsFluorescent in situ hybridization Genomic in situ hybridization Molecular cytogenetics DNA probe Blocking DNA
The authors thank Drs. Bikram S. Gill and Bernd Friebe (Kansas State University, Manhattan, KS, USA) for providing the original protocols. We also thank Drs. Xueyong Zhang (Chinese Academy of Agricultural Sciences, Beijing, China), Qi Zheng (Chinese Academy of Sciences, Beijing, China), Lili Qi (USDA-ARS, Fargo, ND, USA), and Jiming Jiang (University of Wisconsin, Madison, WI, USA) for the instruction and discussion of the procedure. Dr. Jiming Jiang kindly provided the plasmids of 5S rDNA and 45S rDNA. This work was supported by the USDA-ARS CRIS Project No. 3060-520-037-00D. USDA is an equal opportunity provider and employer.
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