20 Strand-Specific Fluorescence in Situ Hybridization: CO-FISH and COD-FISH
265
Thus, single-stranded probes of these sequences will hybridize to only one
chromatid, as shown in Fig. Ib. Repetitive sequences that are present in
both directions within the human genome will hybridize to both chromatids.
The COD-FISH method uses knowledge of the structural organization
of most eukaryotic telomeres to determine the direction of DNA sequences hybridized to slides prepared using the CO-FISH method. The
eukaryotic telomere sequence is a tandem repeat, (TTAGGG)n> with the
C-rich sequence at the 5' end and the G-rich sequence at the 3' end of
each chromosomal DNA strand. Co-hybridization of a single-stranded telomere sequence probe with a single-stranded probe of an interstitial DNA
sequence to CO-FISH prepared chromosomes allows determination of the
strand direction of the interstitial sequence. For example, in Fig. Ic the Crich strand of the telomere sequence probe hybridizes to the G-rich 3' end
of each chromatid. The antisense strand of an interstitial DNA sequence
probe hybridizes to the chromatid with the telomere signal on the short
arm of the chromosome. This information is used to determine the short
arm-to-Iong arm chromosome direction of the sense strand of the interstitial sequence (see Meyne and Goodwin, 1995).
Because multiple, sequential hybridizations can be done without significant degradation of chromosome morphology, the CO-FISH method is
also useful for preparing single-stranded target DNA for hybridization
with denatured double-stranded probes. However, a double-stranded
probe will always hybridize to both chromatids, as each strand of the
probe will bond with its complementary site.
Materials
- Cells in culture and appropriate growth reagents and equipment
- 103
M bromodeoxyuridine stock solution in water. Aliquots of this
solution can be kept frozen for several months, but must be protected
from direct light at all times.
- Phosphate buffered saline
- Colcemid
- 75 mM potassium chloride hypotonic solution
- Methanol
- Acetic Acid
265
Thus, single-stranded probes of these sequences will hybridize to only one
chromatid, as shown in Fig. Ib. Repetitive sequences that are present in
both directions within the human genome will hybridize to both chromatids.
The COD-FISH method uses knowledge of the structural organization
of most eukaryotic telomeres to determine the direction of DNA sequences hybridized to slides prepared using the CO-FISH method. The
eukaryotic telomere sequence is a tandem repeat, (TTAGGG)n> with the
C-rich sequence at the 5' end and the G-rich sequence at the 3' end of
each chromosomal DNA strand. Co-hybridization of a single-stranded telomere sequence probe with a single-stranded probe of an interstitial DNA
sequence to CO-FISH prepared chromosomes allows determination of the
strand direction of the interstitial sequence. For example, in Fig. Ic the Crich strand of the telomere sequence probe hybridizes to the G-rich 3' end
of each chromatid. The antisense strand of an interstitial DNA sequence
probe hybridizes to the chromatid with the telomere signal on the short
arm of the chromosome. This information is used to determine the short
arm-to-Iong arm chromosome direction of the sense strand of the interstitial sequence (see Meyne and Goodwin, 1995).
Because multiple, sequential hybridizations can be done without significant degradation of chromosome morphology, the CO-FISH method is
also useful for preparing single-stranded target DNA for hybridization
with denatured double-stranded probes. However, a double-stranded
probe will always hybridize to both chromatids, as each strand of the
probe will bond with its complementary site.
Materials
- Cells in culture and appropriate growth reagents and equipment
- 103
M bromodeoxyuridine stock solution in water. Aliquots of this
solution can be kept frozen for several months, but must be protected
from direct light at all times.
- Phosphate buffered saline
- Colcemid
- 75 mM potassium chloride hypotonic solution
- Methanol
- Acetic Acid