In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei

Joan H. M. Knoll1, Peter Lichter2

1 Children's Mercy Hospital, University of Missouri‐Kansas City School of Medicine, Kansas City, 2 Deutsches Krebsforschungszentrum, Heidelberg, Germany
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.3
DOI:  10.1002/0471142905.hg0403s45
Online Posting Date:  May, 2005
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Abstract

In situ hybridization is used to determine the chromosomal map location and the relative order of genes and DNA sequences within a chromosomal band. It can also be used to detect aneuploidy, gene amplification, and subtle chromosomal rearrangements. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in the first basic protocol. Two support protocols are provided to amplify weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described in alternate protocols. Nonisotopic labeling of DNA probes by nick translation is described in a support protocol. The order of closely spaced FISH probes along chromosomes in interphase nuclei can be determined. A basic protocol for isotopic in situ hybridization (IISH) with 3H is provided followed by a support protocol for preparation of autoradiographic emulsion.

     
 
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Table of Contents

  • Basic Protocol 1: Fluorescence in Situ Hybridization to Metaphase Chromosomes
  • Amplification of Hybridization Signals
  • Support Protocol 1: Amplification of Biotinylated Signals
  • Support Protocol 2: Amplification of Signals from Digoxigenin‐Labeled Probes
  • Enzymatic Detection of Nonisotopically Labeled Probes
  • Alternate Protocol 1: Enzymatic Detection Using Horseradish Peroxidase
  • Alternate Protocol 2: Enzymatic Detection Using Alkaline Phosphatase
  • Support Protocol 3: Biotin and Digoxigenin Labeling of Probes by Nick Translation
  • Alternate Protocol 3: Ordering of Sequences, Detection of Subtle Abnormalities, and Examination of Allele‐Specific Replication in Interphase Nuclei by FISH
  • Basic Protocol 2: Isotopic in Situ Hybridization with 3H‐Labeled DNA Probes
  • Support Protocol 4: Preparation of Emulsion for Autoradiography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescence in Situ Hybridization to Metaphase Chromosomes

  Materials
  • Slide or coverslip containing metaphase human chromosomes (unit 4.1)
  • Denaturation solution: 70% (v/v) deionized formamide (see recipe)/2× SSC (pH 7.0; appendix 2D), 70°C
  • 70% (ice‐cold), 80%, 95%, and 100% ethanol (prepare 70% and 80% from 95% ethanol)
  • 20 to 150 ng nonisotopically labeled DNA probe: single‐copy, locus specific chromosome‐paint, or repetitive‐element Vysis or Cytocell (see protocol 6)
  • Cot‐1 DNA (Invitrogen)
  • Deionized formamide (see recipe; American Bioanalytical or Sigma)
  • Master hybridization mix (see recipe)
  • 50% (v/v) formamide (not deionized)/2× SSC ( appendix 2D)
  • 2×, 1×, and 4× SSC, pH 7.0 ( appendix 2D)
  • Biotin detection solution or digoxigenin detection solution or biotin/digoxigenin detection solution (see reciperecipes)
  • 0.1% (v/v) Triton X‐100/4× SSC
  • DAPI or propidium iodide staining solution (see reciperecipes)
  • Appropriate antifade mounting medium (see recipe)
  • 72°, 39°, and 42°C water baths
  • Phase‐contrast microscope
  • 22‐mm2 coverslips (plastic and glass)
  • Para film
  • Moist chamber (Fig. )
  • Slide box with desiccant (‐VWR International)
  • Nail polish
  • Fluorescence microscope with epi‐illumination and filter set(s) appropriate for fluorochrome(s) used (Table 97.80.4711) including dual‐band pass filter (fluorescein/Texas red) or triple‐band pass filter (fluorescein/Texas red/DAPI; Omega Optical or Chroma Technology)
  • Ektar‐1000 or Ektachrome‐400 color film or Technical Pan 2415 black‐ and‐white film (Kodak) or computerized imaging system
CAUTION: Formamide, DAPI, and propidium iodide are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Amplification of Biotinylated Signals

  • 1 to 3 µg/ml biotinylated anti‐avidin antibodies (Vector Laboratories) in 4× SSC/ 1% (w/v) BSA (fraction V)
  • Biotin amplification solution: 2 to 5 µg/ml fluorescein‐avidin DCS (Vector Laboratories) in 4× SSC/1% (w/v) BSA (fraction V)
NOTE: This procedure should be performed with minimum exposure to ambient light.

Support Protocol 2: Amplification of Signals from Digoxigenin‐Labeled Probes

  • 10 µg/ml Fab fragment of sheep anti‐digoxigenin (Roche Diagnostics) in 4× SSC/1% (w/v) BSA (fraction V)
  • Digoxigenin amplification solution: 3.5 to 7.0 µg/ml fluorescein‐conjugated rabbit anti–sheep IgG (Sigma) in 4× SSC/1% (w/v) BSA (fraction V)
NOTE: This procedure should be performed with minimum exposure to ambient light.

Alternate Protocol 1: Enzymatic Detection Using Horseradish Peroxidase

  • Blocking solution: 1% (w/v) BSA in PBS ( appendix 2D)
  • Streptavidin solution (see recipe)
  • 0.1% (v/v) Tween 20/PBS ( appendix 2D), 42°C
  • Biotinylated horseradish peroxidase (HRPO) solution (see recipe)
  • DAB substrate solution: 500 µg/ml 3,3′‐diaminobenzidine tetrahydrochloride (DAB) in PBS ( appendix 2D), prepare fresh
  • 3% H 2O 2
  • PBS ( appendix 2D)
  • 90% (v/v) glycerol or appropriate antifade mounting medium (see recipe)
  • 24 × 60–mm coverslips
  • 42°C shaking water bath
CAUTION: DAB is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: This procedure must be performed in the dark with minimum exposure to ambient light.

Alternate Protocol 2: Enzymatic Detection Using Alkaline Phosphatase

  • Blocking solution: 1% (w/v) BSA/PBS ( appendix 2D)
  • Streptavidin solution (see recipe)
  • 0.1% (v/v) Tween 20/PBS ( appendix 2D), 42°C
  • Biotinylated alkaline phosphatase (AP) solution (see recipe)
  • Alkaline phosphatase buffer, pH 9.5 (see recipe), 42°C
  • NBT/BCIP substrate solution (see recipe)
  • PBS ( appendix 2D)
  • 90% glycerol or appropriate antifade mounting medium (see recipe)
  • 24 × 60–mm coverslips
  • 42°C shaking water bath
NOTE: This procedure must be performed in the dark with minimum exposure to ambient light.

Support Protocol 3: Biotin and Digoxigenin Labeling of Probes by Nick Translation

  Materials
  • Nick translation kit (e.g., Roche Diagnostics) or equivalent materials:
    • 10× nick translation buffer (see recipe)
    • 1 µg probe DNA (e.g., PCR amplicons, cloned inserts in plasmids, phage, cosmids, P1, or single YACs)
    • 1 mM biotin‐16‐dUTP or 1 mM digoxigenin‐11‐dUTP (Roche Diagnostics)
    • 1 mg/ml DNase I (Worthington or appendix 2D)
    • 5 U/ml DNA polymerase I, endonuclease‐free (Roche Diagnostics)
  • 0.5 M EDTA
  • 10% (w/v) SDS
  • 10 mg/ml sonicated salmon sperm DNA (see recipe)
  • 3 M sodium acetate, pH 5.2
  • 95% and 70% (v/v) ethanol, ice‐cold
  • 15° and 65°C water baths
  • 65° heating block
  • Additional reagents and equipment for agarose electrophoresis (unit 2.7)

Alternate Protocol 3: Ordering of Sequences, Detection of Subtle Abnormalities, and Examination of Allele‐Specific Replication in Interphase Nuclei by FISH

  Materials
  • Slide containing metaphase chromosomes (unit 4.1)
  • Denaturation solution: 70% (v/v) deionized formamide (see recipe)/2× SSC (pH 7.0; appendix 2D), 70°C
  • 70% (ice‐cold), 80%, 95% (v/v), and 100% ethanol (prepare 70% and 80% from 95% ethanol)
  • 500 ng/ml 3H‐labeled probe DNA, labeled by random oligonucleotide–primed synthesis ( appendix 3E), 107 cpm/µg DNA
  • 10× SSCP (see recipe)
  • 20% (w/v) dextran sulfate (500,000 mol. wt.; Pharmacia Biotech) in deionized formamide, autoclaved
  • 10 mg/ml sonicated salmon sperm DNA (see recipe)
  • 50% (v/v) formamide (not deionized)/2× SSC (pH 7.0; appendix 2D), 39°C
  • 2× SSC (pH 7.0; appendix 2D), 39°C
  • Diluted NTB‐2 autoradiographic emulsion (see protocol 9)
  • Desiccant (VWR International)
  • Kodak D19 developer diluted 1:1 with H 2O, 15°C
  • Kodak fixer, 15°C
  • 0.5% (w/v) quinacrine dihydrochloride (see recipe)
  • McIlvaine buffer, pH 5.5 (see recipe)
  • Sucrose mounting medium: 60% (w/v) sucrose in McIlvaine buffer, pH 5.5
  • 72°, 39°, 45°, and 15°C water baths
  • 24 × 60–mm no. 1 coverslips
  • Rubber cement
  • 50‐ml centrifuge tube
  • Safelight with Wratten series 2 filter
  • Moist chamber (Fig. )
  • Black light‐tight slide box (e.g., Becton Dickinson Primary Care or VWR Scientific)
  • Light‐tight slide‐dryer box with built‐in fan
  • Black electrical tape
  • Nail polish
  • Fluorescence microscope with filter set for quinacrine fluorescence (Table 4.3.1)
  • Technical Pan 2415 film (Kodak)
CAUTION: Formamide, 3H, and quinacrine are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol 2: Isotopic in Situ Hybridization with 3H‐Labeled DNA Probes

  Materials
  • NTB‐2 autoradiographic emulsion (Kodak; available wrapped in lead)
  • 50‐ml centrifuge tubes
  • 45°C water bath
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Figures

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Literature Cited

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