An improved method for generating BAC DNA suitable for FISH.
Fluorescence in situ hybridization (FISH) is commonly used to identify chromosomal aberrations such as translocations, deletions, duplications, gene fusions, and aneuploidies. It relies on the hybridization of fluorescently labeled DNA probes onto denatured metaphase chromosomes or interphase nuclei. These probes are often generated from DNA sequences cloned within bacterial artificial chromosomes (BACs). Growing these BACs in adequate amounts for FISH can be demanding. We describe FISH performed with bacteriophage Phi29 DNA polymerase amplified BAC DNA. Generating this material required significantly smaller cultures and less time than standard methods. The FISH results obtained were comparable with those obtained from standard BAC DNA. We believe this method of BAC DNA generation is useful for the entire FISH community as it improves considerably on prior methods.
Department of Genetics, State University of New York at Stony Brook, Stony Brook, NY 11794-8691, USA. jasmin.roohi@hsc.stonybrook.edu
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J
Roohi
M
M
Cammer
C
C
Montagna
E
E
Hatchwell
D017103:Bacillus Phages / Q000201:enzymology; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D002877:Chromosomes, Human / Q000235:genetics; D003582:Cytogenetics / Q000379:methods; D004274:DNA, Recombinant / Q000096:biosynthesis* / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D020558:GTP Phosphohydrolases / Q000235:genetics; D018734:Genes, erbB-2; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D058112:Septins
D004274:DNA, Recombinant; D004259:DNA-Directed DNA Polymerase; D020558:GTP Phosphohydrolases; C474315:SEPTIN9 protein, human; D058112:Septins