Strand-Specific Fluorescence in Situ Hybridization: CO-FISH and COD-FISH

Abstract

The ability to prepare single-stranded target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methods require functionally single-stranded DNAs at the beginning of the hybridization step to allow nucleotide base pairing between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probe and target DNAs to induce a temporary separation of the DNA strands. The strand-specific FISH method involves selective removal of one of the strands from the DNA helixes within metaphase chromosomes and/or interphase nuclei. The result, for the purposes of hybridization, is single-stranded target DNA. Single-stranded probes can then be hybridized to the single-stranded target DNA without the denaturation step, resulting in strand-specific hybridization. In the context of this chapter, “single-stranded probe” refers to nucleotide sequences without complementary strands present, not denatured double-stranded probes.