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JULIANNE MEYNE, SUSAN M. BAILEY, and EDWIN H. GOODWIN
arm-to-Iong arm direction of DNA sequences along chromosomes. This
variation of the method is called COD-FISH (Chromosome Orientation
and Direction-FISH; Meyne and Goodwin, 1995). Multiple color and sequential hybridizations are easily accomplished on slides prepared using
the CO-FISH method. Because no denaturation step is required the chromosomes maintain their morphology, even after several hybridizations.
Outline
Figure 1 summarizes the strategy of our approach for obtaining singlestranded DNA for CO-FISH. The procedure is effective for both metaphase
chromosomes and interphase DNA. Cultured cells are grown for one
round of replication in medium containing the nucleotide analogue bromodeoxyuridine (BrdU). Because of the semiconservative nature of DNA
synthesis, BrdU will be substituted for thymidine at many sites within the
newly replicated DNA strands. Thus, each newly replicated chromosome
contains sister chromatids with singly substituted complementary DNA
strands. That is, BrdU partially replaces thymidine in one DNA strand
of each chromatid and the substitution takes place in opposite strands
of the two sister chromatids (Fig. la). At this point, the cells are prepared
for microscopy. Any of the standard metaphase chromosome preparation
methods or whole cell fixation methods compatible with FISH can be used
for CO-FISH. Cells can also be grown directly on coverglasses or microscope slides and fixed in place.
At this point, the newly replicated DNA strands are removed. The prepared slides are stained with the fluorescent DNA-binding dye Hoechst
33258 and then exposed to long-wave ultraviolet light. The UV exposure
induces numerous strand breaks that preferentially occur at BrdU substitution sites. The nicked DNA strands produced by this treatment serve as a
substrate for digestion by exonuclease III. This enzyme excises nucleotides
by starting at the sites of nicks and proceeding along the DNA strand. This
process effectively removes the BrdU substituted strand from each chromatid, while leaving the unsubstituted strand essentially intact. It is important to note that not only is each chromatid now single-stranded, but
the single DNA strands remaining in the sister chromatids are complementary.
Strand-specific hybridization of CO-FISH slides requires that the probe
must also be single-stranded. The complement of DNA sequences present
as single copies in the genome or of repetitive sequences arranged as a
head-to-tail tandem array will only be present on one of the DNA strands.
JULIANNE MEYNE, SUSAN M. BAILEY, and EDWIN H. GOODWIN
arm-to-Iong arm direction of DNA sequences along chromosomes. This
variation of the method is called COD-FISH (Chromosome Orientation
and Direction-FISH; Meyne and Goodwin, 1995). Multiple color and sequential hybridizations are easily accomplished on slides prepared using
the CO-FISH method. Because no denaturation step is required the chromosomes maintain their morphology, even after several hybridizations.
Outline
Figure 1 summarizes the strategy of our approach for obtaining singlestranded DNA for CO-FISH. The procedure is effective for both metaphase
chromosomes and interphase DNA. Cultured cells are grown for one
round of replication in medium containing the nucleotide analogue bromodeoxyuridine (BrdU). Because of the semiconservative nature of DNA
synthesis, BrdU will be substituted for thymidine at many sites within the
newly replicated DNA strands. Thus, each newly replicated chromosome
contains sister chromatids with singly substituted complementary DNA
strands. That is, BrdU partially replaces thymidine in one DNA strand
of each chromatid and the substitution takes place in opposite strands
of the two sister chromatids (Fig. la). At this point, the cells are prepared
for microscopy. Any of the standard metaphase chromosome preparation
methods or whole cell fixation methods compatible with FISH can be used
for CO-FISH. Cells can also be grown directly on coverglasses or microscope slides and fixed in place.
At this point, the newly replicated DNA strands are removed. The prepared slides are stained with the fluorescent DNA-binding dye Hoechst
33258 and then exposed to long-wave ultraviolet light. The UV exposure
induces numerous strand breaks that preferentially occur at BrdU substitution sites. The nicked DNA strands produced by this treatment serve as a
substrate for digestion by exonuclease III. This enzyme excises nucleotides
by starting at the sites of nicks and proceeding along the DNA strand. This
process effectively removes the BrdU substituted strand from each chromatid, while leaving the unsubstituted strand essentially intact. It is important to note that not only is each chromatid now single-stranded, but
the single DNA strands remaining in the sister chromatids are complementary.
Strand-specific hybridization of CO-FISH slides requires that the probe
must also be single-stranded. The complement of DNA sequences present
as single copies in the genome or of repetitive sequences arranged as a
head-to-tail tandem array will only be present on one of the DNA strands.