Chapter 20
PROTOCOL
Strand-Specific Fluorescence in Situ Hybridization:
CO-FISH and COD-FISH
JULIANNE MEYNE, SUSAN M. BAILEY, and EDWIN H. GOODWIN
, Introduction
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.
Using the CO-FISH method, single-stranded probes of repetitive DNA
sequences arranged in head-to-tail tandem arrays along the chromosome
or sequences present as single copies in the genome will hybridize to only
one chromatid of a metaphase chromosome. Probes of DNA sequences,
such as Alu, that are present in both directions on each DNA strand will
hybridize to both chromatids. Thus, the method can be used to determine
~ Julianne Meyne, Los Alamos National Laboratory, Bioscience Division, MS-M888,
Los Alamos, NM, 87545, USA (phone +505-667-2697; fax +505-667-2891;
e-mail co-fish@telomere.lanl.gov)
Susan M. Bailey, Los Alamos National Laboratory, Bioscience Division, MS-M888, Los
Alamos, NM, 87545, USA
Edwin H. Goodwin, Los Alamos National Laboratory, Bioscience Division, MS-M888,
Los Alamos, NM, 87545, USA