Advanced Preparative Techniques to Establish Probes for Molecular Cytogenetics

Jan Stap1, Jacob A. Aten1, D. Lillington2, A. Shelling2, B.D. Young2

1 University of Amsterdam, Amsterdam, 2 St. Bartholomew's Hospital School of Medicine, London
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.6
DOI:  10.1002/0471142956.cy0806s05
Online Posting Date:  May, 2001
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Abstract

The methods covered in this unit include flow cytometry of metaphase chromosomes, chromosome dissection, and the DOP‐PCR amplification methods for reverse chromosome painting. Successful application in these areas requires care and attention to methodological details, and this unit is particularly comprehensive.

Keywords: molecular cytogenetics; chromosomes; flow cytometry; microdissection; metaphase; cytogenetics.

     
 
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Table of Contents

  • Basic Protocol 1: Preparative Techniques for Flow Cytometric Analysis of Chromosomes
  • Support Protocol 1: Preparation of Flow Sorter for Chromosome Analysis
  • Basic Protocol 2: Chromosome Harvesting, Banding, and Microdissection
  • Support Protocol 2: Preparing Microneedles for Microdissection
  • Basic Protocol 3: Degenerate Oligonucleotide–Primed PCR (DOP‐PCR)
  • Support Protocol 3: FISH Analysis of PCR Products
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparative Techniques for Flow Cytometric Analysis of Chromosomes

  Materials
  • Human cell culture
  • 10 µg/ml Colcemid (demecolcine; Sigma; store ≥8 weeks at 4°C)
  • 37.5 mM KCl
  • Polyamine solutions 1 and 2 (see recipe)
  • 1 M MgSO 4
  • 500 µg/ml 4′,6‐diamidino‐2‐phenylindole (DAPI) or 1 mg/ml propidium iodide (PI)
  • 5 mg/ml (100×) chromomycin A3 (Sigma) in ethanol (store ≥6 weeks at –20°C)
  • 100 µg/ml (100×) Hoechst 33258 in H 2O (store ≥6 weeks at –20°C)
  • 1 M sodium citrate
  • Latex beads for aligning lasers (see protocol 2 and manufacturer's instructions)
  • Collection buffer for sorted chromosomes (composition appropriate for subsequent analysis) containing at least 0.1% (w/v) bovine serum albumin (BSA, fraction V; Sigma)
  • Glycerol or 100% ethanol
  • Tabletop centrifuge
  • Syringes with 0.7 × 40–mm (22‐G) needles
  • Fluorescence microscope
  • Flow cytometer/cell sorter (e.g., Becton Dickinson FACStar Plus or FACSVantage) with two argon lasers and the following filters:
  •  For Hoechst 33258 detection (green fluorescence): 480‐nm short‐pass filter and 380‐ or 390‐nm long‐pass filter to block scattered UV laser light
  •  For chromomycin A3 detection (blue fluorescence): 490‐nm long‐pass filter
  • Siliconized tubes (e.g., 1.7‐ml Costar microcentrifuge tubes, no. 3207) or BSA‐coated microcentrifuge tubes (see recipe)
  • Cytocentrifugation system: e.g., Leif bucket (Leif, )
  • Microscope slides (precleaned with 1:1 ethanol/ether)
  • Additional materials for cell culture ( appendix 3B) and for preparing and aligning flow sorter (see protocol 2 and manufacturer's instructions)
CAUTION: All DNA stains—e.g., PI, DAPI, chromomycin A3, and Hoechst 33258—are suspected to be toxic and mutagenic and should be handled with care. See unit 3.3 regarding aerosol containment during flow sorting.NOTE: All solutions and equipment coming into contact with cells must be sterile and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Preparation of Flow Sorter for Chromosome Analysis

  • 1:20 sodium hypochlorite or cleaning solution recommended by sorter manufacturer (e.g., FACSafe and FACSRinse; Becton Dickinson)
  • 70% ethanol (∼0.5 liter needed for sterilizing tubing of cell sorter)
  • Sterile distilled water
  • 0.9% NaCl, sterile
  • 1 µg/ml Hoechst 33258 in 1× phosphate‐buffered saline (PBS; see recipe), sterile
  • Latex beads for aligning the lasers: 1.0 ± 0.01 µm yellow/green Fluoresbrite microspheres for aligning 457‐nm laser and 1.0 ± 0.01 µm bright blue Fluoresbrite microspheres for aligning the UV laser (Polysciences)

Basic Protocol 2: Chromosome Harvesting, Banding, and Microdissection

  Materials
  • Human cell culture
  • 10 µg/ml Colcemid (demecolcine; Sigma; store ≥8 weeks at 4°C)
  • 0.075 M KCl, 37°C
  • 70% ethanol, ice cold
  • Fixative: 3 parts methanol/1 part glacial acetic acid (ice cold)
  • Trypsin solution: 1 ml Bacto Trypsin (Difco) reconstituted in 40 ml of 0.9% NaCl
  • 0.9% NaCl
  • Gurr buffer pH 6.8 (Bio/medical Specialties)
  • 1 part Leishman's stain (e.g., Baxter) in 4 parts Gurr buffer, pH 6.8 (prepare fresh)
  • Taq DNA polymerase buffer
  • Coverslips, cleaned with methanol and kept in sterile container in freezer
  • Zeiss Axiovert inverted microscope with 16× eyepieces, 4× or 6× objective, 63× dry lens objective, and gliding and rotating stage
  • Remote‐controlled micromanipulator (Zeiss) and mounts for micromanipulator
  • Vibration‐free table
  • Microdissection needles (see protocol 4)
NOTE: All solutions and equipment coming into contact with cells must be sterile and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Preparing Microneedles for Microdissection

  Materials
  • 70% ethanol
  • 1‐ or 1.5‐mm‐diameter borosilicate glass rods or capillary tubes
  • Microelectrode puller (e.g., Campden Instruments or David Kopf Instruments)
  • Grinder (e.g., Narashige) with lens system
  • Microneedle holders (custom made)
  • Microoven

Basic Protocol 3: Degenerate Oligonucleotide–Primed PCR (DOP‐PCR)

  Materials
  • Microdissected chromosomal fragments in 5 µl Taq DNA polymerase buffer (see protocol 3)
  • 50 mM MgCl 2
  • 5 U/ml Taq DNA polymerase
  • 5 mM 4dNTP mix: 5 mM each of dATP, dCTP, dGTP, and dTTP
  • 20 µM DOP‐PCR primer (CCGACTCGAGNNNNNNATGTGG, where N = A, C, G, or T and the underlined bases represent an XhoI restriction site)
  • Sterile H 2O
  • Mineral oil
  • Automated thermal cycler

Support Protocol 3: FISH Analysis of PCR Products

  Materials
  • DNA from DOP‐PCR (see protocol 5)
  • Cot‐1 DNA (Life Technologies)
  • 3 M sodium acetate, pH 5.6 (unit 8.3)
  • 100% ethanol, ice‐cold
  • Hybridization buffer (see recipe)
  • Cells containing chromosomes for hybridization
  • RNase A solution (see recipe)
  • Denaturing solution (see recipe; prepare fresh)
  • 20× SSC, pH 5.3, and 2× SSC, pH 7 ( appendix 2A)
  • Dehydration series: 70%, 95%, and 100% ethanol, ice‐cold
  • Rubber cement
  • Formamide wash solution (see recipe)
  • 4× SSCT (see recipe)
  • Blocking solution (see recipe; prepare fresh)
  • 5 µg/ml fluorescein isothiocyanate–conjugated avidin (avidin‐FITC; Sigma or Vector Laboratories; store up to 6 months at −20°C)
  • 5 µg/ml biotinylated anti–avidin D antibodies (Vector Laboratories; store up to 6 months at −20°C)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Citifluor/PI (see recipe)
  • DNA purification system: Geneclean kit (Bio 101) or Promega Wizard PCR Preps DNA Purification Kit
  • 42°, 75°, and 90°C water baths
  • Microscope slides
  • 32 × 22–mm coverslips
  • Moist chamber: 1‐liter beaker containing paper towels moistened with 2× SSC ( appendix 2A), covered with aluminum foil
  • Coplin jars
  • Confocal or fluorescence microscope (see Chapter 2)
  • Additional reagents and equipment for labeling of DNA by nick translation and purification of labeled probes for FISH (unit 8.3) and spreading of metaphase chromosomes on slides for FISH (unit 8.2)
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Figures

Videos

Literature Cited

   Aten, J.A., Buys, C.H.C.M., van der Veen, A.Y., Mesa, J.R., Yu, L.C., Gray, J.W., Osinga, J., and Stap, J. 1987. Stabilization of chromosomes by DNA intercalators for flow karyotyping and identification by banding of isolated chromosomes. Histochemistry 87:359‐366.
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   Boschman, G.A., Buys, C.H., van de Veen, A.Y., Rens, W., Osinga, J., Slater, R.M., and Aten, J.A. 1993. Identification of a tumor marker chromosome by flow sorting, DNA amplification in vitro and in situ hybridization of the amplified product. Genes Chromosomes Cancer 6:10‐16.
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   Cotter, F.E., Lillington, D.M., Hampton, G., Riddle, P., Nasipuri, S., Gibbons, B., and Young, B.D. 1991. Gene mapping by microdissection and enzymatic amplification: Heterogeneity in leukemia‐associated breakpoints on chromosome 11. Genes Chromosomes Cancer 3:8‐15.
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   Cremer, C., Rappold, G., Gray, J.W., Muller, C.R., and Ropers, H.H. 1984. Preparative dual‐beam sorting of the human Y chromosome and in situ hybridization of cloned DNA probes. Cytometry 5:572‐579.
   Dracopoli, N.C., Haines, J.L., Korf, B.R., Moir, D.T., Morton, C.C., Seidman, C.E., Seidman, J.G., and Smith, D.R. (eds.). 1998. Current Protocols in Human Genetics. John Wiley & Sons, New York.
   Edstrom, J.‐E., Kaiser, R., and Rohmes, D. 1987. Microcloning of mammalian metaphase chromosomes. Methods Enzymol. 151:503‐516.
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   Kao, F.T. 1993. Microdissection and microcloning of human chromosome regions in genome and genetic disease analysis. BioEssays 15:141‐146.
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   Rens, W., Boschman, G.A., Hoovers, J.M., Manders, E.M.M., Slater, R.M., Stap, J., and Aten, J.A. 1994. Flow cytometric detection of chromosome abnormalities by measuring centromeric index, DNA content and DNA base composition. Anal. Cell. Pathol. 6:359‐375.
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   Sillar, R. and Young, B.D. 1981. A new method for the preparation of metaphase chromosomes for flow analysis. J. Histochem. Cytochem. 29:74‐78.
   Suijkerbuijk, R.J., Matthopoulos, D., Kearney, L., Monard, S., Dhut, S., Cotter, F.E., Herbergs, J., Geurts van Kessel, A., and Young, B.D. 1992. Fluorescence in situ identification of human marker chromosomes using flow sorting and Alu element‐mediated PCR. Genomics 13:355‐362.
   Telenius, H., Carter, N.P., Bebb, C.E., Nordenskjold, M., Ponder, B.A.J., and Tunnacliffe, A. 1992a. Degenerate oligonucleotide–primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13:718‐725.
   Telenius, H., Pelmear, A.H., Tunnacliffe, A., Carter, N.P., Behmel, A., Ferguson‐Smith, M.A., Nordenskjold, M., Pfragner, R., and Ponder, B.A.J. 1992b. Cytogenetic analysis by chromosome painting using DOP‐PCR amplified flow‐sorted chromosomes. Genes Chromosomes Cancer 4:257‐263.
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Key References
   Sillar and Young, 1981. See above.
  Preparative technique for chromosome sorting.
   Telenius et al., 1992a. See above.
  DOP‐PCR method.
   Lüdecke et al., 1998. See above.
  Microdissection and cloning.
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