92
MORIMASA MATSUTA AND MAYUMI MATS UTA
• Poor metaphase morphology
- Repeat the hybridization on a new specimen using one of
the following. 1. Decrease the hybridization temperature by
2°C. 2. Decrease the denaturation temperature. 3. Fix the
slides in 4% paraformaldehyde/PB for 10 mins, wash the
slides in distilled water several times, and then air-dry.
Subprotocol 4
Comparative Genomic Hybridization (CGH)
Outline
Principle of CGH (see also Figure 8):
1. Preparation of normal metaphase spreads that serve as targets
for the hybridization.
2. Isolation of high-molecular-weight genomic DNA from tumor specimens.
3. Labeling of the tumor and normal DNA with different fluorochromes.
4. In situ hybridization of the labeled DNAs to a normal metaphase, washing the unbound DNA fragments.
5. Fluorescence microscopy to visualize color ratio differences
along metaphase chromosomes.
6. Acquisition and display of multi-color digital images.
7. Quantitation of copy number difference by generating green
to red fluorescence intensity ratio profiles for all chromosomes in a metaphase spread.
8. Combining the profiles from several metaphases to improve
signal to noise ratio.
9. Interpretation of ratio changes and verification of results by
control experiments.
MORIMASA MATSUTA AND MAYUMI MATS UTA
• Poor metaphase morphology
- Repeat the hybridization on a new specimen using one of
the following. 1. Decrease the hybridization temperature by
2°C. 2. Decrease the denaturation temperature. 3. Fix the
slides in 4% paraformaldehyde/PB for 10 mins, wash the
slides in distilled water several times, and then air-dry.
Subprotocol 4
Comparative Genomic Hybridization (CGH)
Outline
Principle of CGH (see also Figure 8):
1. Preparation of normal metaphase spreads that serve as targets
for the hybridization.
2. Isolation of high-molecular-weight genomic DNA from tumor specimens.
3. Labeling of the tumor and normal DNA with different fluorochromes.
4. In situ hybridization of the labeled DNAs to a normal metaphase, washing the unbound DNA fragments.
5. Fluorescence microscopy to visualize color ratio differences
along metaphase chromosomes.
6. Acquisition and display of multi-color digital images.
7. Quantitation of copy number difference by generating green
to red fluorescence intensity ratio profiles for all chromosomes in a metaphase spread.
8. Combining the profiles from several metaphases to improve
signal to noise ratio.
9. Interpretation of ratio changes and verification of results by
control experiments.