Traditional Banding of Chromosomes for Cytogenetic Analysis

Jane Bayani1, Jeremy A. Squire1

1 University of Toronto, Ontario, null
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.3
DOI:  10.1002/0471143030.cb2203s23
Online Posting Date:  September, 2004
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Abstract

Traditional banding of metaphase chromosomes allows identification of individual chromosomes and detection of gross chromosomal anomalies and abnormal chromosome structures. Giemsa (G) banding is the older and more familiar nonfluorescent technique that produces characteristic banding patterns. DAPI , a fluorescent DNA stain, produces banding patterns similar to those of G‐banding and is much simpler to perform.

Keywords: G‐banding; DAPI banding; metaphase spreads

     
 
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Table of Contents

  • Basic Protocol 1: Giemsa Banding (G‐Banding) of Metaphase Chromosomes
  • Alternate Protocol 1: 4′‐6‐Diamidino‐2‐Phenylindole (DAPI) Staining of Metaphase Chromosomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Giemsa Banding (G‐Banding) of Metaphase Chromosomes

  Materials
  • Metaphase slide preparation made 1 to 2 days before banding (unit 22.2preparation of cytogenetic specimens from tissue samples)
  • Trypsin working solution (see recipe)
  • 1% (v/v) fetal bovine serum (FBS): mix 1 ml FBS with 99 ml H 2O
  • Gurr's buffer, pH 6.8: dissolve 1 Gurr's buffer table (Bio/medical Specialties) in 1 liter sterile H 2O
  • Giemsa stain working solution (see recipe)
  • 55° to 60°C drying oven
  • Forceps
  • Coplin jars
  • Phase‐contrast microscope

Alternate Protocol 1: 4′‐6‐Diamidino‐2‐Phenylindole (DAPI) Staining of Metaphase Chromosomes

  Materials
  • Metaphase slide preparation (unit 22.2preparation of cytogenetic specimens from tissue samples)
  • DAPI/antifade medium (see recipe)
  • Glass coverslips
  • Fluorescent microscope with DAPI filters
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Figures

  •   FigureFigure 22.3.1 Images of G‐banded metaphase spreads and karyotypes from human, mouse and rat tumors. For each panel, a G‐banded metaphase spread and its karyotype has been shown for tumors derived from (A) human, (B) mouse, and (C) rat. All spreads show banding patterns that are well defined with few or no overlapping chromosomes.
  •   FigureFigure 22.3.2 DAPI staining of a prostate cell line subjected to a chemotherapeutic agent and 4 Gy radiation. Shown in panel A, labeled “i,” is a DAPI image of a prostate cell line subjected to mitomycin C treatment. Structural aberrations, e.g., radial structures, are labeled “ii.”. Also in panel A is the inverted DAPI image (labeled “iii”) demonstrating that DAPI banding provides similar banding patterns as G‐banding, but at a lower resolution. Panel B illustrates the same prostate cell line subjected to 4 Gy of ionizing radiation, showing complex structural aberrations. Other aberrations, including DNA fragmentation, chromatid breaks, and dicentrics, can be identified. Arrows indicate multiradial chromosomal structures as shown enlarged in (ii).

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Literature Cited

Literature Cited
   Barch, M.J. 1991. The ACT Cytogenetics Laboratory Manual, 2nd Ed. Raven Press, New York.
   Ellis, N.A., Proytcheva, M., Sanz, M.M., Ye, T.Z., and German, J. 1999. Transfection of BLM into cultured bloom syndrome cells reduces the sister‐chromatid exchange rate toward normal. Am. J. Hum. Genet. 65:1368‐1374.
   Gebhart, E. 1981. Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing. Hum. Genet. 58:235‐254.
   Mitelman, F. 1995. ISCN. 1995 An International System for Human Cytogenetic Nomenclature 1995. S. Karger, Basel, Switzerland.
   Sumner, A.T., Evans, H.J., and Buckland, R.A. 1971. New technique for distinguishing between human chromosomes. Nat. New Biol. 7:232:31‐32.
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