High‐Resolution FISH Analysis

Henry H.Q. Heng1, Bradford Windle2, Lap‐Chee Tsui3

1 Center for Molecular Medicine and Genetics Wayne State University School of Medicine, Detroit, Michigan, 2 Virginia Commonwealth University, Richmond, Virginia, 3 University of Hong Kong, Hong Kong
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.5
DOI:  10.1002/0471142905.hg0405s44
Online Posting Date:  February, 2005
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Abstract

Map order, orientation, and gap or overlap distance of closely linked DNA probes may be determined using fluorescent hybridization to decondensed DNA. The linear arrangement of released chromatin fibers not only simplifies the task of gene ordering, but also provides higher resolution with probes separated by greater distances than can be achieved in FISH with intact interphase nuclei. The of this unit describes an alkaline lysis procedure for generating free chromatin from cultured cells for FISH analysis. A support protocol describes an empirical approach to optimize conditions for preparation of free chromatin. An provides a method for producing free chromatin from cultured lymphocytes with drug treatment. The , high‐resolution FISH mapping with free chromatin, is a modification of the method used for FISH mapping of interphase nuclei.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Chromatin Fiber with Alkaline Buffer
  • Support Protocol 1: Optimization of Chromatin Fiber Preparation
  • Alternate Protocol 1: Preparation of Chromatin Fibers from Lymphocytes by Drug Treatment
  • Preparation of DNA Fibers
  • Basic Protocol 2: Preparation of DNA Fibers Using SDS Lysis and Gravity
  • Alternate Protocol 2: Preparation of DNA Fibers Using Alkaline Treatment Plus Mechanical Pulling
  • Basic Protocol 3: Fish Detection with Stretched Chromatin and DNA Fibers
  • Support Protocol 2: Biotin and Digoxigenin Labeling of Fish Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Chromatin Fiber with Alkaline Buffer

  Materials
  • Fibroblast culture or 10 ml fresh human peripheral or cord blood
  • Fibroblast or lymphocyte culture medium (see recipe; with and without serum for fibroblast medium)
  • Trypsin/EDTA solution (Invitrogen)
  • Alkaline buffer (see recipe)
  • Fixative: 3:1 (v/v) methanol/glacial acetic acid (prepare fresh)
  • 60‐mm tissue culture plates
  • 25‐cm2 tissue culture flasks
  • 15‐ml screw‐cap polystyrene tubes
  • IEC HN‐S centrifuge and 958 (or equivalent) rotor
  • Microscope slides, chilled 5 to 10 min on ice
  • Phase‐contrast microscope
  • Slide box
  • Additional reagents and equipment for tissue culture and trypsinization of cells ( appendix 3G)

Support Protocol 1: Optimization of Chromatin Fiber Preparation

  • 4% Giemsa stain: 2 ml Giemsa stain (Fisher)/48 ml Gurr, pH 6.8 (BDH), prepared fresh

Alternate Protocol 1: Preparation of Chromatin Fibers from Lymphocytes by Drug Treatment

  • 3 ml fresh human peripheral or cord blood
  • 5 mg/ml m‐AMSA (see recipe) or 10 mg/ml ethidium bromide ( appendix 2D)
  • 0.4% (w/v) KCl
CAUTION: m‐AMSA and ethidium bromide are mutagens and should be handled with care.

Basic Protocol 2: Preparation of DNA Fibers Using SDS Lysis and Gravity

  Materials
  • Cultured cells
  • Phosphate‐buffered saline (PBS; appendix 2D)
  • Lysis buffer (see recipe)
  • Fixative: 3:1 (v/v) methanol/glacial acetic acid (prepare fresh)
  • N 2 gas
  • 25 × 75 × 1–mm glass microscope slides
  • Moist chamber (Fig. )
  • Heat‐sealable bags
  • Drierite, 8‐mesh (Fisher)

Alternate Protocol 2: Preparation of DNA Fibers Using Alkaline Treatment Plus Mechanical Pulling

  Materials
  • Cultured cells
  • 0.4% (w/v) KCl
  • Fixative: 3:1 (v/v) methanol/glacial acetic acid (prepare fresh)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Alkaline solution: 5:2 (v/v) 0.07 M NaOH/100% ethanol (prepare fresh)
  • Methanol
  • 70%, 95%, and 100% ethanol
  • 15‐ml centrifuge tubes
  • IEC HN‐S centrifuge and 958 (or equivalent) rotor
  • 25 × 75 × 1–mm glass microscope slides
  • Coverslips

Basic Protocol 3: Fish Detection with Stretched Chromatin and DNA Fibers

  Materials
  • Glass slides containing streams of stretched DNA or stretched chromatin fiber (see protocol 1 or protocol 42, or protocol 5)
  • 100 µg/ml RNase: dilute 2 mg/ml RNase stock (DNase‐free; appendix 2D) in 2× SSC; prepare fresh
  • 4× and 2× SSC ( appendix 2D)
  • 70% (v/v) deionized formamide/2× SSC, 70°C
  • 70% (v/v) ethanol, ice‐cold
  • 100% and 90% (v/v) ethanol, room temperature
  • Labeled probes (see protocol 7)
  • Sonicated genomic DNA (100 to 1000 bp; see recipe) orC 0t 1 DNA (e.g., COT‐1 DNA, Invitrogen)
  • Hybridization buffer (see recipe)
  • Rubber cement
  • 50% (v/v) nondeionized formamide/2× SSC
  • Preavidin block solution (see recipe)
  • 5 µg/ml fluorescein‐avidin DCS (Vector) in 4× SSC/1% (w/v) BSA
  • 4× SSC/0.1% (v/v) Triton X‐100
  • PN buffer (see recipe)
  • NGS/PN solution: 4% (v/v) normal goat serum (NGS, Vector) in PN buffer (store 1‐ml aliquots at 4°C)
  • 5 µg/ml biotinylated anti‐avidin D antibody (Vector) in NGS/PN solution
  • 25 µg/ml mouse anti‐digoxigenin antibody (Roche) in NGS/PN solution
  • 25 µg/ml digoxigenin‐labeled polyvalent anti‐mouse Ig F(ab′) 2 fragment (Roche) in NGS/PN solution
  • 25 µg/ml rhodamine‐conjugated anti‐digoxigenin Fab fragment (Roche) in NGS/PN solution
  • Vectashield antifade mounting medium (Vector)
  • 22 × 40–mm glass coverslips, no. 1
  • Polyethylene Coplin jars
  • Moist chamber (Fig. )
  • 45° and 75°C water baths
  • Rubber cement
  • Epifluorescence microscope (unit 4.4) with triple‐band‐pass filter (or set of single‐band‐pass filters for FITC and rhodamine) and 100× oil objective
  • CCD camera (e.g., Photometrics CoolSNAP)
  • Image analysis software (e.g., Photometrics RS Image)

Support Protocol 2: Biotin and Digoxigenin Labeling of Fish Probes

  Materials
  • Probe DNA to be labeled
  • Nick translation DNA labeling kit, e.g., BioNick (Invitrogen) or Boehringer DIG nick translation mix (Roche Applied Sciences); alternatively, prepare nick translation mix in‐house (see recipe)
  • Stop buffer: 0.5 M EDTA, pH 8.0 ( appendix 2D)
  • TE buffer, pH 7.5 ( appendix 2D)
  • Sonicated salmon sperm DNA (Invitrogen; see unit 4.3 for sonication)
  • 3 M sodium acetate ( appendix 2D)
  • 70% and 100% ethanol
  • 15°C water bath
  • Nick column (Amersham Pharmacia Biotech)
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Figures

Videos

Literature Cited

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Key References
   Heng et al., 1992. See above.
  The first paper introducing the concept of high‐ resolution fiber FISH.
   Parra and Windle, 1993. See above.
  Shows a variety of data generated with the DNA fiber mapping protocol
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