6 In Situ Hybridization for DNA: Fluorescent Probe
95
- Cot-l DNA (lO)1g, from Gibco-BRL or Vysis)
- Normal male or female human genomic DNA
Procedure
1. Nick translation of DNA samples for comparative genomic
hybridization. In a micro centrifuge tube, mix:
- l)1g tumor DNA in 38)11 double distilled water
5)1110 x A4 mixture
1)11 (-lnmol) FITC-12-dUTP (DuPont, Boston, MA)
5)11 enzyme mixture containing SNA polymerase 1(-2 U)
and DNAse 1(-200 pg) (Gibco BRL, Gaithersburg, MO)
1)11 (-10 U) DNA polymerase I (Promega, Madison, WI)For labeling of normal references DNA, FITC is substituted
with Tex-Red-5-dUTP (DuPont).
2. Incubate 45-60 mins at 15°C in a water bath.
3. Stop reaction by a 10 mins incubation at 70°C
Note: Run a double-stranded 1 % agarose gel to check the probe
fragment distribution. Size range should be about 500-2,000 bp.
If the probe fragments are longer, increase the incubation time or
the amount of enzyme mixture and if shorter, decrease the incubation time or the amount of enzyme mixture.
4. Preparation of target metaphase slides: Mark the area of the
slide containing metaphases with a diamond pen (18 mm
square)
5. Denature normal lymphocyte metaphase slides for 3 mins at
74°C in a denaturation solution (70% formamide/2xSSC)
6. Dehydrate in a sequence of 70, 85, and 100% ethanol, 2mins
each
7. Incubate slides in a proteinase K solution (O.l)1g/ml in 20 mM
Tris-HC1!2mM CaCh, pH 7.5) for 7.5 mins at room temperature
8. Dehydrate as above
9. Preparation of hybridization mix:
Mix in a micro centrifuge tube:
Comparative
genomic hybridization
95
- Cot-l DNA (lO)1g, from Gibco-BRL or Vysis)
- Normal male or female human genomic DNA
Procedure
1. Nick translation of DNA samples for comparative genomic
hybridization. In a micro centrifuge tube, mix:
- l)1g tumor DNA in 38)11 double distilled water
5)1110 x A4 mixture
1)11 (-lnmol) FITC-12-dUTP (DuPont, Boston, MA)
5)11 enzyme mixture containing SNA polymerase 1(-2 U)
and DNAse 1(-200 pg) (Gibco BRL, Gaithersburg, MO)
1)11 (-10 U) DNA polymerase I (Promega, Madison, WI)For labeling of normal references DNA, FITC is substituted
with Tex-Red-5-dUTP (DuPont).
2. Incubate 45-60 mins at 15°C in a water bath.
3. Stop reaction by a 10 mins incubation at 70°C
Note: Run a double-stranded 1 % agarose gel to check the probe
fragment distribution. Size range should be about 500-2,000 bp.
If the probe fragments are longer, increase the incubation time or
the amount of enzyme mixture and if shorter, decrease the incubation time or the amount of enzyme mixture.
4. Preparation of target metaphase slides: Mark the area of the
slide containing metaphases with a diamond pen (18 mm
square)
5. Denature normal lymphocyte metaphase slides for 3 mins at
74°C in a denaturation solution (70% formamide/2xSSC)
6. Dehydrate in a sequence of 70, 85, and 100% ethanol, 2mins
each
7. Incubate slides in a proteinase K solution (O.l)1g/ml in 20 mM
Tris-HC1!2mM CaCh, pH 7.5) for 7.5 mins at room temperature
8. Dehydrate as above
9. Preparation of hybridization mix:
Mix in a micro centrifuge tube:
Comparative
genomic hybridization