Genetics and Genomics > Cancer Genetics

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Molecular Analysis of Gene Amplification in Tumors

Jonathon C. Wasson1,  Garrett M. Brodeur1

1Washington University School of Medicine, St. Louis, Missouri

Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 10.5
DOI: 
10.1002/0471142905.hg1005s02
Online Posting Date: 
May, 2001
GO TO THE FULL TEXT:
PDF or HTML at Wiley Online Library
Are you the author of this protocol? Login or register and return to this page.

Abstract

Detection of gene amplification in human cancer cells has both clinical and biological importance. Amplified genes can be classified in one of two categoriesas oncogenes or as genes conferring drug resistance. Both types of gene amplification may alter clinical management of the patient. The basic protocol describes preparation and quantitation of DNA from tumor tissue and the use of conventional Southern blot hybridization analysis to detect and quantify gene amplification. The first alternate protocol provides an approach to quickly screen tumor samples for gene amplification using slot blot hybridization analysis. The second alternate protocol describes the use of the polymerase chain reaction (PCR) for analyzing tumors that may be difficult to analyze because of degradation or limited amounts of DNA. A Support Protocol explains the proper methods for obtaining, processing, storing, and shipping tumor tissue for DNA analysis.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • Basic Protocol: Detection of Gene Amplification by Southern Blot Hybridization Analysis
  • Alternate Protocol 1: Detection of Gene Amplification by Slot Blot Hybridization Analysis
  • Alternate Protocol 2: Detection of Gene Amplification by Differential PCR
  • Support Protocol: Obtaining and Processing Tumor Tissue
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol: Detection of Gene Amplification by Southern Blot Hybridization Analysis

 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.
  • Tumor tissue (Alternate Protocol 1)
  • Stratagene DNA Extraction Kit (Stratagene) or equivalent:
    • DNA extraction solution: 50 mM Tris. Cl (pH 8.0)/20 mM EDTA (pH 8.0)/2% (w/v) SDS
  • 225 mg/ml pronase
  • NaCl extraction solution: saturated NaCl solution
  • 10 mg/ml RNase
  • 100% and 70% ethanol, ice cold
  • TE buffer, pH 8.0
  • 10, 100, 250, and 500 µg/ml calf thymus DNA standards (see recipe)
  • Capillary assay solution (see recipe), prepared just before use
  • 1× TNE buffer (see recipe)
  • 1 M spermidine (see recipe)
  • Single-copy control DNA
  • Amplified control DNA
  • 10 U/µl restriction enzyme and appropriate 10× buffer
  • 1 mg/ml bovine serum albumin (BSA)
  • 4 M NaCl
  • 6× gel loading buffer (appendix 2A)
  • Agarose
  • 1× TBE buffer (appendix 2A)
  • 10 mg/ml ethidium bromide
  • Denaturing solution: 0.5 NaOH/1 M NaCl, prepared fresh and equilibrated 30 min
  • Neutralizing solution (see recipe), prepared fresh
  • 6× SSC (appendix 2A)
  • Quik-Hyb solution (Stratagene)
  • 10 mg/ml herring sperm DNA, sonicated and boiled (see recipe)
    • Double-stranded DNA probe, labeled with [-32P]dCTP by random oligonucleotide priming to a specific activity ³1 × 109 cpm/µg DNA and spin-column-purified (appendix 3E)
  • Wash solution I: 2× SSC/0.2% (w/v) SDS, prepared fresh, 55°C
  • Wash solution II: 0.5× SSC/0.2% (w/v) SDS, prepared fresh, 55°C
  • Wash solution III: 0.2×SSC/0.2% (w/v) SDS, prepared fresh, 55°C
  • 7.5-ml ground-glass tissue homogenizer or equivalent
  • 30- and 50-ml centrifuge tubes
  • Sorvall centrifuge and JS-5.2 rotor (or equivalent)
  • Large-bore pipets
  • Vacuum pump apparatus (creating ³30 mm Hg) or Speedvac
  • 2- or 5-ml O-ring screw-cap tubes
  • Rocker platform
  • Mini-fluorometer (Hoefer)
  • 0.5- and 1.5-ml microcentrifuge tubes
  • Nitrocellulose membrane
  • 80°C vacuum oven or UV cross-linker (e.g., Stratalinker, Stratagene)
  • Heat-sealable polyethylene bags
  • 65°C shaking water bath
  • X-AR autoradiographic film
  • Additional reagents and equipment for agarose gel electrophoresis and Southern blotting (unit 2.7)

Alternate Protocol 1: Detection of Gene Amplification by Slot Blot Hybridization Analysis

 Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.
  • 3.0 M NaOH, filter sterilized
  • 2.0 M ammonium acetate, pH 7.0, filter sterilized
  • Repeating pipettor (Brinkmann)
  • Dot or slot blotting manifold (e.g., Bio-Dot SF, Bio-Rad; or Minifold 2, Schleicher & Schuell)
  • Whatman 3MM paper

Alternate Protocol 2: Detection of Gene Amplification by Differential PCR

 Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • 10× Taq DNA polymerase buffer: 500 mM KCl/100 mM Tris-Cl, pH 8.3 (Store £18 months at –20°C)
  • 25 mM MgCl2
  • 1.25 mM 4dNTP mix (appendix 2A)
  • 20 µM target and control oligonucleotide primers, + and – strand sequences of each
  • 5 U/µl Taq DNA polymerase
  • Mineral oil (not needed if thermal cycler has a heated lid)
  • 2% (w/v) agarose in TBE buffer (appendix 2A)
  • Low-molecular-weight DNA size markers (e.g., BioMarker Low, Bioventures)
  • 0.5-ml microcentrifuge tube
  • Automated thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Support Protocol: Obtaining and Processing Tumor Tissue

 Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.
  • Tumor tissue
  • Liquid N2
  • Cryotubes
Looking for materials?  Suppliers Guide
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  • Figure 10.5.1
    Southern blot analysis for N-myc amplification. (A) Southern blot results of five neuroblastomas (lanes 1 to 5) with a normal N-myc copy number, compared to a control cell line (NGP; Brodeur et al., 1977; Schwab et al., 1983, 1984) with 150-fold amplification (lane 6). (B) Southern blot results with two tumors without amplification (lanes 1 and 2) and four tumors with varying degrees of N-myc amplification: 150-fold (lane 3); 50-fold (lane 4), 5-fold (lane 5), and 100-fold (lane 6).

    View Image
  • Figure 10.5.2
    Slot blot analysis for N-myc amplification. Column A shows six serial dilutions of a control cell line (NGP) with 150-fold amplification of N-myc (row 1). Serial dilutions show ~75-, ~40- ~20-, ~10-, ~5-, and ~2.5-fold amplification (rows 2 to 7 respectively); row 8 has normal DNA as a control. Columns B and C show slot blot results of 18 tumors. B-6 has ~80-fold amplification, B-9 has ~15-fold amplification, and C-6 has ~30-fold amplification, based on quantitative densitometry compared to the standards (column A). All other samples have nonamplified DNA.

    View Image
  • Figure 10.5.3
    PCR analysis for N-myc amplification. Lanes 1 to 3 contain PCR products from neuroblastomas that had a normal N-myc copy number (based on Southern blotting). Lanes 4-6 have PCR products from tumors or cell lines with N-myc amplification (lanes 4 and 5: ~100-fold amplification; lane 6: ~150-fold amplification). Amplification is seen primarily by a progressive decrease in the intensity of the control band (alpha-fetoprotein, or AFP), although there is some increase in the N-myc band as well.

    View Image

Literature Cited

 Literature Cited
    Alitalo, K. and Schwab, M. 1986. Oncogene amplification in tumor cells. Adv. Cancer Res 47:235-281.
    Bonilla, M., Ramirez, M., Lopez-Cueto, J., and Gariglio, P. 1988. In vivo amplification and rearrangement of c-myc oncogene in human breast tumors. J. Nat. Cancer Inst. 80:665-671.
    Brodeur, G.M. and Fong, C.T. 1989. Molecular biologyand genetics of human neuroblastoma. Cancer Genet. Cytogenet 41:153-174.
    Brodeur, G.M. and Seeger, R.C. 1986. Gene amplification in human neuroblastomas: Basic mechanisms and clinical implications. Cancer Genet. Cytogenet. 19:101-111.
    Brodeur, G.M., Sekhon, G.S., and Goldstein, M.N. 1977. Chromosomal aberrations in human neuroblastomas. Cancer 40:2256-2263.
    Brodeur, G.M., Seeger, R.C., Schwab, M., Varmus, H.E., and Bishop, J.M. 1984. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224:1121-1124.
    Brodeur, G.M., Hayes, F.A., Green, A.A., Casper, J.T., Wasson, J., Wallach, S., and Seeger, R.C. 1987. Consistent N-myc copy number in simultaneous or consecutive neuroblastoma samples from sixty individual patients. Cancer Res. 47:4248-4253.
    Brodeur, G.M., Azar, C., Brother, M., Hiemstra, J., Kaufman, B., Marshall, H., Moley, J., Nakagawara, A., Saylors, R., Scavarda, N., Schneider, S., Wasson, J., White, P., Seeger, R., Look, T., and Castleberry, R. 1992. Neuroblastoma: Effect of genetic factors on prognosis and treatment. Cancer 70:1685-1694.
    Carman, M.D., Schornagel, J.H., Rivest, R.S., Srimatkandada, S., Portlock, C.S., Duffy, T., and Bertino, J.R. 1984. Resistance to methotrexate due to gene amplification in a patient with acute leukemia. J. Clin. Oncol. 2:16-20.
    Crabbe, D.C.G., Peters, J., and Seeger, R.C. 1992. Identification of neuroblastomas with genomic amplification of MYCN by polymerase chain reaction (PCR). Proc. Am. Assoc. Cancer Res. 33:254.
    Curt, G.A., Carney, D.N., Cowan, K.H., Jolivet, J., Bailey, B.D., Kao-, C.S., Shan, C.S., Minna, J.D., and Chabner, B.A. 1983. Unstable methotrexate resistance in human small-cell carcinoma associated with double minute chromosomes. New Engl. J. Med. 308:199-202.
    Escot, C., Theillet, C., Lidereau, R., Spyratos, F., Champeme, M.-H., Gest, J., and Callahan, R. 1986. Genetic alteration of the c-myc protooncogene (MYC) in human primary breast carcinomas. Proc. Natl. Acad. Sci. U.S.A. 83:4834-4838.
    Frye, R.A., Benz, C.C., and Liu, E. 1989. Detection of amplified oncogenes by differential polymerase chain reaction. Oncogene 4:1153-1157.
    Horns, R.C.J., Dower, W.J., and Schimke, R.T. 1984. Gene amplification in a leukemia patient treated with methotrexate. J. Clin. Oncol. 2:2-7.
    Johnson, B.E., Ihde, D.C., Makuch, R.W., Gazdar, A.F., Carney, D.N., Oie, H., Russell, E., Nau, M.M., and Minna, J.D. 1987. Myc family oncogene amplification in tumor cell lines established from small cell lung cancer patients and its relationship to clinical status and course. J. Clin. Invest. 79:1629-1634.
    Kallioniemi, O.-P., Kallioniemi, A., Kurisu, W., Thor, A., Chen, L.-C., Smith, H.S., Waldman, F.M., Pinkel, D., and Gray, J.W. 1992a. ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 89:5321-5325.
    Kallioniemi, A., Kallioniemi, O.-P., Sudar, D., Rutovitz, D., Gray, J.W., Waldman, F., and Pinkel, D. 1992b. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818-821.
    Kozbor, D. and Croce, C.M. 1984. Amplification of the c-myc oncogene in one of five human breast carcinoma cell lines. Cancer Res. 44:438-441.
    Little, C.D., Nau, M.M., Carney, D.N., Gazdar, A.F., and Minna, J.D. 1983. Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature 306:194-196.
    Look, A.T., Hayes, F.A., Shuster, J.J., Douglass, E.C., Castleberry, R.P., Bowman, L.C., Smith, E.I., and Brodeur, G.M. 1991. Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: A Pediatric Oncology Group study. J. Clin. Oncol. 9:581-591.
    Merkel, D.E., Fuqua, S.A.W., Tandon, A.K., Hill, S.M., Buzdar, A.U., and McGuire, W.L. 1989. Electrophoretic analysis of 248 clinical breast cancer specimens for P-glycoprotein overexpression or gene amplification. J. Clin. Oncol 7:1129-1136.
    Nakagawara, A., Ikeda, K., Tsuda, T., and Higashi, K. 1987. N-myc oncogene amplification and prognostic factors of neuroblastoma in children. J. Pediatr. Surg. 22:895-898.
    Nau, M.M., Brooks, B.J., Battey, J.E.S., Gazdar, A.F., Kirsch, I.R., McBride, O.W., Bertness, V., Hollis, G.F., and Minna, J.D. 1985. L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature 318:69-73.
    Nau, M.M., Brooks, B.J.J., Carney, D.N., Gazdar, A.F., Battey, J.F., Sausville, E.A., and Minna, J.D. 1986. Human small-cell lung cancers show amplification and expression of the N-myc gene. Proc. Natl. Acad. Sci. U.S.A. 83:1092-1096.
    Saksela, K., Bergy, J., Lehto, V.P., Nillson, K., and Alitalo, K. 1985. Amplification of the c-myc oncogene in a subpopulation of human small cell lung cancer. Cancer Res. 45:1823-1827.
    Schwab, M., Alitalo, K., Klempnauer, K.H., Varmus, H.E., Bishop, J.M., Gilbert, F., Brodeur, G., Goldstein, M., and Trent, J.M. 1983. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumor. Nature 305:245-248.
    Schwab, M., Varmus, H.E., Bishop, J.M., Grzeschik, K.H., Naylor, S.L., Sakaguchi, A.Y., Brodeur, G., and Trent, J. 1984. Chromosome localization in normal human cells and neurobastomas of a gene related to c-myc. Nature 308:288-291.
    Seeger, R.C., Brodeur, G.M., Sather, H., Dalton, A., Siegel, S.E., Wong, K.Y., and Hammond, D. 1985. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. New Engl. J. Med. 313:1111-1116.
    Shapiro, D.N., Valentine, M.B., Rowe, S.T., Sinclair, A.E., Sublett, J.E., Roberts, W.M., and Look, A.T. 1993. Detection of N-myc gene amplification by fluorescence in-situ hybridization: Diagnostic utility for neuroblastoma. Am. J. Path 142:1339-1346.
    Slamon, D.J., Clark, G.M., Wong, S.G., Jevin, W.J., Ullrich, A., and McGuire, W.L. 1987. Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177-182.
    Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A., Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G., Udove, J., Ullrich, A., and Press, M.F. 1989. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707-712.
    Slavc, I., Ellenbogen, R., Jung, W.-H., Vawter, G.F., Kretschmar, C., Grier, H., and Korf, B.R. 1990. Myc gene amplification and expression in primary human neuroblastoma. Cancer Res. 50:1459-1463.
    Theillet, C., LeRoy, X., DeLapeyiere, O., Grosgeorges, J., Adnane, J., Reynaud, S.D., Simony-Lafontaine, J., Goldfarb, M.E.C., Birnbaum, D., and Gaudray, P. 1989. Amplification of FGF-related genes in human tumors: Possible involvement of HST in breast carcinomas. Oncogene 4:915-922.
    Thenne, S., Fury, J., Todd, S., Sakaguchi, A.Y., and Naylor, S.L. 1991. PCR primers for human chromosomes: Reagents for the rapid analysis of somatic cell hybrids. Genomics 9:511-516.
    Trent, J.M., Buick, R.N., Olson, S., Horns, R.C.J., and Schimke, R.T. 1984. Cytologic evidence for gene amplification in methotrexate-resistant cells obtained from a patient with ovarian adenocarcinoma. J. Clin. Oncol. 2:8-15.
    Wong, A.J., Ruppert, J.M., Eggleston, J., Hamilton, S.R., Baylin, S.B., and Vogelstein, B. 1986. Gene amplification of c-myc and N-myc in small cell carcinoma of the lung. Science 233:461-464.
 Key Reference
    Kellems, R.E., ed. 1993. Gene Amplification in Mammalian Cells. Marcel Dekker, New York.

A recent, comprehensive review of gene amplification in mammalian cells, including amplification of oncogenes and drug resistance genes.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.