Generation of Inexpensive, Highly Labeled Probes for Fluorescence In Situ Hybridization (FISH)

• Published in: STAR protocols Vol. 1; no. 1; p. 100006
• Main Authors: Sharma, Rahul, Meister, Peter
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.06.2020; Elsevier
• Subjects: Animals; Humans; In Situ Hybridization, Fluorescence - methods; Mice; Molecular Probes - chemistry; Molecular Probes - metabolism; Polymerase Chain Reaction - methods; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2019.100006

DNA-FISH remains the method of choice to visualize genomic regions in situ ranging from a single locus to entire chromosomes. Current methods to generate probes rely on expensive kits that vary in labeling efficiency and are limited by the size and/or amount of starting material and by the choice of fluorophores. Here we describe a protocol to prepare inexpensive ($20) DNA-FISH probes using an isothermal polymerase, incorporating labeled nucleotides while amplifying minute amounts of any template (PCR fragments/BAC/YAC/fosmids). For complete details on the use and execution of this protocol, please refer to Grosmaire et al. (2019) and Sharma et al. (2014). [Display omitted] •Direct incorporation of modified nucleotides during isothermal template amplification•Adaptable to the size, amount, type of template DNA, and choice of fluorophores•Inexpensive probes and easily scalable from single locus to multiple chromosomes•Method consistently yields good quantity of highly labeled probes (∼1 dye/100 bases) DNA-FISH remains the method of choice to visualize genomic regions in situ ranging from a single locus to entire chromosomes. Current methods to generate probes rely on expensive kits that vary in labeling efficiency and are limited by the size and/or amount of starting material and by the choice of fluorophores. Here we describe a protocol to prepare inexpensive ($20) DNA-FISH probes using an isothermal polymerase, incorporating labeled nucleotides while amplifying minute amounts of any template (PCR fragments/BAC/YAC/fosmids).