Analysis of Sister‐Chromatid Exchanges

James German1, Becky Alhadeff1

1 New York Blood Center, New York, New York
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.6
DOI:  10.1002/0471142905.hg0806s02
Online Posting Date:  May, 2001
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Abstract

Two requirements for the cytogenetic analysis of sister‐chromatid exchanges (SCEs) in somatic cells are (1) a population of actively proliferating cells that will provide an adequate number of metaphases and (2) sister chromatids that in some way are differentially labeled or stained in the metaphases. SCEs can be recognized as abrupt discontinuities in the staining patterns of the two chromatids of a metaphase chromosome at what appear to be identical sites, with reciprocal switching from one chromatid to its sister. This protocol uses phytohemagglutinin (PHA)‐stimulated cultures of blood lymphocytes as a source of proliferating cells. The cells are incubated with the thymidine analog BrdU. Slides prepared from fixed cells with BrdU‐substituted chromosomes are treated with Hoechst 33258, exposed to light and heat, and then Giemsa‐stained to produce differentially stained chromosomes. The chromatids with bifilar substitution exhibit a lighter purple stain than their unifilarly substituted sister chromatids.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Sister‐Chromatid Exchanges in Mammalian Metaphase Chromosomes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Sister‐Chromatid Exchanges in Mammalian Metaphase Chromosomes

  Materials
    For recipes, see in this unit (unless cross‐referenced to another unit); for common stock solutions, see appendix 2D 2D; for suppliers, see suppliers appendix.
  • Sterile heparinized blood
  • Mitogen‐containing culture medium: complete RPMI/15% FBS ( appendix 3G) supplemented with 2% (v/v) phytohemagglutinin A (PHA; GIBCO/BRL)
  • recipe2 mM 5‐bromo‐2'‐deoxyuridine (BrdU) stock solution (see recipe)
  • 10 µg/ml Colcemid (e.g., GIBCO/BRL; store at 4°C)
  • Hypotonic solution: 0.075 M KCl, 37°C (store ≤2 weeks at 4°C)
  • Fixative: 3:1 (v/v) absolute methanol/glacial acetic acid (freshly prepared)
  • recipe250 µg/ml Hoechst 33258 stock solution (see recipe)
  • Giemsa stain (Harleco Modified Azure Blend Type, Baxter Scientific; Fisher; Gurr Improved R66, Bio/medical Specialties)
  • Gurr buffer, pH 6.8, from tablets (Bio/medical Specialties)
  • 25‐cm2 tissue culture flasks
  • 15‐ml conical glass or polypropylene centrifuge tubes
  • Standard tabletop centrifuge
  • Polyethylene transfer pipets
  • Precleaned microscope slides, wiped with soft tissue paper and dipped into room‐temperature water just before use
  • Black plastic microscope slide box (VWR Scientific)
  • 24 × 50–mm coverslips, wiped with soft tissue paper just before use
  • Coplin jars
  • Lamp equipped with a 60‐W incandescent bulb
  • 37° and 60°C water baths
  • Rubber cement
  • Scalpel blades
  • Mounting medium (e.g., Permount)
  • Kodak TMax 100 Professional Film
NOTE: Use disposable latex gloves when handling BrdU, Hoechst 33258 dye, and Giemsa stain. BrdU and Hoechst 33258 dye are light‐sensitive materials. Wrap all containers, including tissue culture flasks, centrifuge tubes, and Coplin jars, in foil. Use indirect lighting (e.g., light from an adjacent room) or a shielded low‐wattage light bulb when working with these reagents.NOTE: All incubations are performed in a 37°C, 5% CO 2 humidified incubator unless otherwise specified.NOTE: Sterile culture technique should be employed in steps and .
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Figures

Videos

Literature Cited

Literature Cited
   Block, A.M.W. 1982. Sister chromatid exchange methodology. In Sister Chromatid Exchange (A.A. Sandberg, ed.) pp. 13‐32. Alan R. Liss, New York.
   Chaganti, R.S.K., Schonberg, S., and German, J. 1974. A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 71:4508‐4512.
   German, J., Schonberg, S., Louie, E., and Chaganti, R.S.K. 1977. Bloom's syndrome. IV. Sister chromatid exchanges in lymphocytes. Am. J. Hum. Genet. 29:248‐255.
   Gratzner, H.G., Pollack, A., Ingram, D.J., and Leif, R.C. 1976. Deoxyribonucleic acid replication in single cells and chromosomes by immunological techniques. J. Histochem. Cytochem. 24:34‐39.
   Gutierrez, C., Hernandez, P., and Lopez‐Saez, J.F. 1984. BrdUrd‐independent and BrdUrd‐dependent SCEs as components of SCE yields: Implications for their cellular significance. In Sister Chromatid Exchanges (R.R. Tice and A. Hollaender, eds.) pp. 83‐90. Plenum, New York.
   Latt, S.A. 1973. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. Natl. Acad. Sci. U.S.A. 70:3395‐3399.
   Latt, S.A., Shreck, R.R., D'Andrea, A., Kaiser, T.N., Schlesinger, F., Lester, S., and Sakai, K. 1984. Detection, significance, and mechanism of sister chromatid formation: Past experiments, current concepts, future challenges. In Sister Chromatid Exchanges (R.R. Tice and A. Hollaender, eds.) pp. 11‐40. Plenum, New York.
   Perry, P. and Wolff, S. 1974. New Giemsa method for the differential staining of sister chromatids. Nature 257:156‐158.
   Ray, J.H. and German, J. 1982. Sister chromatid exchange in the chromosome breakage syndromes. In Sister Chromatid Exchange (A.A. Sandberg, ed.) pp. 553‐577. Alan R. Liss, New York.
  Sandberg, A.A. (ed.) 1982. Sister Chromatid Exchange. Alan R. Liss, New York.
   Taylor, J.H. 1984. A brief history of the discovery of sister chromatid exchanges. In Sister Chromatid Exchange (R.R. Tice and A. Hollaender, eds.) pp. 1‐9. Plenum, New York.
  Tice, R.R. and Hollaender, A. (eds.) 1984. Sister Chromatid Exchanges. Plenum, New York.
   Wolff, S. 1982. Sister Chromatid Exchange. John Wiley & Sons, New York.
   Wolff, S. and Perry, P. 1974. Differential Giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography. Chromosoma 48:341‐353.
   Zakharov, A.F. 1982. Historical aspects of sister chromatid exchange. In Sister Chromatid Exchange (A.A. Sandberg, ed.) pp. 1‐12. Alan R. Liss, New York.
   Zakharov, A.F. and Egolina, N.A. 1972. Differential spiralization along mammalian mitotic chromosomes. I. BUdR‐revealed differentiation in Chinese hamster chromosomes. Chromosoma 38:341‐365.
Key References
   Chaganti et al., 1974. See above.
  Reports the increased SCE frequency in Bloom's syndrome.
   Latt, 1973. See above.
  Describes visualization by fluorescence microscopy of differential BrdU incorporation in sister chromatids stained with Hoechst 33258.
   Perry and Wolff, 1974. See above.
  Describes procedure for permanent differential staining of sister chromatids that have been exposed to Hoechst 33258.
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