Probe Labeling and Fluorescence In Situ Hybridization

J. Wiegant1, A.K. Raap1

1 Leiden University, Leiden
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.3
DOI:  10.1002/0471142956.cy0803s00
Online Posting Date:  May, 2001
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Abstract

This unit describes in detail basic protocols for probe labeling, denaturing of in situ target DNA, in situ hybridization, and post‐hybridization washes. Support protocols for probe labeling cover probe purification and quality control. Keywords: molecular cytogenetics; FISH; nick translation; random priming; PCR labeling

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Probe Labeling by Nick Translation
  • Basic Protocol 2: Probe Labeling by Random Priming
  • Basic Protocol 3: Probe Labeling by Polymerase Chain Reaction
  • Alternate Protocol 1: Optimized PCR Labeling of Plasmid DNA for FISH
  • Support Protocol 1: Preparation of Purified Probe Stock Solutions
  • Support Protocol 2: Determination of Probe Fragment Size and Estimation of Probe Concentration
  • Support Protocol 3: Determination of Labeling Efficiency by Dot Blot Assay
  • Basic Protocol 4: Combinatorial Labeling for Multicolor FISH
  • Alternate Protocol 2: Ratio‐Labeling for Multicolor FISH
  • Basic Protocol 5: FISH Using Low‐Complexity Probes and No Repeat Suppression
  • Basic Protocol 6: FISH Using High‐Complexity Probes Conditions and Repeat Suppression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Probe Labeling by Nick Translation

  Materials
  • 10× nick translation buffer (see recipe)
  • 0.1 M DTT ( appendix 2A)
  • Nucleotide mixture (see recipe)
  • 1 mM labeled dUTP: biotin‐16‐dUTP or digoxigenin‐11‐dUTP (Boehringer Mannheim) or fluorochrome‐labeled dUTP (Boehringer Mannheim, NEN Life Science Products, or Amersham)
  • 1 µg/µl probe template DNA
  • 10 U/µl DNA polymerase I (Promega)
  • 1 µg/ml DNase I, freshly diluted (see recipe)
  • 15°C water bath
  • Additional reagents and equipment for preparation of labeled probe stock (see protocol 5), determination of probe fragment size (see protocol 6), and checking hapten incorporation by dot blot (see protocol 7)

Basic Protocol 2: Probe Labeling by Random Priming

  Materials
  • Probe template DNA
  • Digoxigenin–, biotin–, or fluorescein–high prime kit (Boehringer Mannheim)
  • Boiling water bath
  • Additional reagents and equipment for preparation of labeled probe stock (see protocol 5), determination of probe fragment size (see protocol 6), and checking hapten incorporation by dot blot (see protocol 7)

Basic Protocol 3: Probe Labeling by Polymerase Chain Reaction

  Materials
  • 10× PCR buffer, pH 8.3 (see recipe)
  • 25 mM MgCl 2
  • 10× PCR‐dig, PCR‐bio, or PCR‐fluorescein labeling mix (see reciperecipes)
  • PCR primers 1 and 2
  • Template DNA
  • 1 U/µl Taq DNA polymerase
  • Mineral oil
  • PCR thermal cycler
  • Additional reagents and equipment for preparation of a labeled probe stock (see protocol 5), determination of probe fragment size (see protocol 6), and determination of labeling efficiency by dot blot (see protocol 7)

Alternate Protocol 1: Optimized PCR Labeling of Plasmid DNA for FISH

  • 10× PCR buffer: see recipe but adjust pH to 9 and add 1% (v/v) Triton X‐100
  • Alternate nucleotide mixture: 2.5 mM dATP, dCTP, and dGTP and 1.5 mM dTTP
  • 0.25 mM biotin‐16‐dUTP (Boehringer Mannheim)
  • 0.25 mM digoxigenin‐11‐dUTP (Boehringer Mannheim)
  • 0.25 mM fluorescein‐dUTP (Boehringer Mannheim, NEN Life Science Products, or Amersham)
  • 1 ng/µl plasmid template DNA
  • 50 pM PCR primers 1 and 2
  • 5 U/µl Taq DNA polymerase (e.g., Promega)

Support Protocol 1: Preparation of Purified Probe Stock Solutions

  Materials
  • Probe labeling reaction mix (see protocol 1, protocol 22, or protocol 33 or protocol 4)
  • 0.5 M EDTA, pH 8 ( appendix 2A)
  • MicroSpin columns (Pharmacia Biotech)
  • 10 mg/ml sonicated and denatured fish sperm DNA (see recipe)
  • 3 M sodium acetate, pH 5.6 (see recipe)
  • 70% and 100% ethanol, −20°C
  • 1 µg/µl C 0t 1 DNA (Life Technologies or Boehringer Mannheim)
  • Hybridization solution I or II (see recipe and Table 8.3.1)
  • Aspirator

Support Protocol 2: Determination of Probe Fragment Size and Estimation of Probe Concentration

  Materials
  • Agarose (e.g., Promega)
  • 50× TAE buffer ( appendix 2A)
  • 10 mg/ml ethidium bromide
  • Probe labeling reaction mix (see protocol 1, protocol 22, or protocol 33 or protocol 4)
  • 6× gel loading buffer ( appendix 2A)
  • DNA molecular size markers, (e.g., 1 kb DNA ladder, Life Technologies)
  • Biotin‐ digoxigenin‐, or fluorescein‐labeled DNA standard (e.g., Boehringer Mannheim)
CAUTION: Ethidium bromide is a mutagen and must be handled carefully.

Support Protocol 3: Determination of Labeling Efficiency by Dot Blot Assay

  Materials
  • Probe labeling reaction mix (see protocol 1)
  • TE buffer ( appendix 2A)
  • Hybond N+ membrane (Amersham)
  • 2× SSC ( appendix 2A)
  • 1 ng/µl biotin‐, digoxigenin‐, or fluorescein‐labeled standard DNA (in TE buffer; Life Technologies or Boehringer Mannheim)
  • TNB buffer (see recipe)
  • AP‐conjugated streptavidin, sheep anti‐digoxigenin, or sheep anti‐fluorescein (e.g., Boehringer Mannheim)
  • TNT buffer (see recipe)
  • NBT/BCIP substrate solution (see recipe)
  • 80°C oven

Basic Protocol 4: Combinatorial Labeling for Multicolor FISH

  Materials
  • Set of probe stock solutions, e.g.:
  •  probe 1: biotin labeled
  •  probe 2: fluorescein labeled
  •  probe 3: rhodamine labeled
  •  probe 4: biotin labeled
  •  probe 4: fluorescein labeled
  •  probe 5: biotin labeled
  •  probe 5: rhodamine labeled
  •  probe 6: fluorescein labeled
  •  probe 6: rhodamine labeled
  •  probe 7: biotin labeled
  •  probe 7: fluorescein labeled
  •  probe 7: rhodamine labeled.

Alternate Protocol 2: Ratio‐Labeling for Multicolor FISH

  Materials
  • Set of probe stock solutions, e.g.:
  •  probe 1: biotin labeled
  •  probe 2: biotin labeled
  •  probe 2: digoxigenin labeled
  •  probe 3: biotin labeled
  •  probe 3: digoxigenin labeled
  •  probe 4: biotin labeled
  •  probe 4: digoxigenin labeled
  •  probe 5: digoxigenin labeled.

Basic Protocol 5: FISH Using Low‐Complexity Probes and No Repeat Suppression

  Materials
  • Probe stock solution (see protocol 5)
  • Hybridization solution I or II (see reciperecipes and Table 8.3.1)
  • Biological sample or microscopic specimen on slide, pretreated for FISH (unit 8.2)
  • Microscopic preparations (unit 8.2)
  • 50% formamide/2× SSC, pH 7
  • 60% formamide/2× SSC, pH 7
  • 0.1× and 2× SSC ( appendix 2A)
  • 70%, 90%, and 100% ethanol
  • TNT buffer (see recipe)
  • Counterstain
  • Mounting medium
  • 18 × 18–mm coverslips
  • 80°C hot plate
  • Schiefferdecker jars (Fisher)
  • Shaking water baths, up to 60°C
  • Additional reagents and equipment for immunocytochemistry (e.g., Watkins, )

Basic Protocol 6: FISH Using High‐Complexity Probes Conditions and Repeat Suppression

  • 70% deionized formamide ( appendix 2A)/2× SSC ( appendix 2A)/50 mM sodium phosphate, pH 7 (see recipe)
  • 24 × 60–mm coverslips
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Figures

Videos

Literature Cited

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   Moore, D., Chory, J., and Ribaudo, R.K. 1994. Isolation and purification of large DNA restriction fragments from agarose gels. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhleds, eds.) pp. 2.6.1‐2.6.12. John Wiley & Sons, New York.
   Nederlof, P.M., Robinson, D., Abuknesha, R., Wiegant, J., Hopman, A.H.N., Tanke, H.J., and Raap, A.K. 1989. Three‐color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10:20‐27.
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Key References
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