Chromosome Microdissection

Paul Meltzer1, Michael Bittner1

1 National Institute for Human Genome Research, Bethesda, Maryland
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.8
DOI:  10.1002/0471142905.hg0408s14
Online Posting Date:  May, 2001
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Abstract

This unit describes chromosome microdissection methodologies that are useful in both clinical and research laboratories. In combination with FISH, region‐specific probes can be made to determine the origin of marker chromosomes. Another application of the microdissection technology is in the generation of regionspecific cDNA libraries, known as microdissection‐mediated cDNA capture.

     
 
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Table of Contents

  • Basic Protocol 1: Microdissection and PCR Amplification of Banded Human Chromosomes
  • Basic Protocol 2: Isolating Region‐Specific cDNAs by Microdissection‐Mediated cDNA Capture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microdissection and PCR Amplification of Banded Human Chromosomes

  Materials
  • Cultured cells
  • recipeCollection buffer (see recipe)
  • Human genomic DNA
  • 10 U/µl topoisomerase I
  • 12 U/µl modified T7 DNA polymerase (e.g., Sequenase, version 2) and enzyme dilution buffer
  • recipePCR mix (see recipe)
  • Low‐molecular‐weight DNA size markers
  • recipePCR mix (see recipe) containing biotin nucleotide mix or Spectrum Orange nucleotide mix ( appendix 2D)
  • 3 M sodium acetate, pH 5.2
  • 100% ethanol, ice‐cold
  • 100 mM Tris⋅Cl (pH 7.5)/1 mM EDTA
  • Glass slides or 22 × 60–mm glass coverslips
  • 1.2‐mm glass capillary tubes (World Precision Instruments)
  • Pipet puller
  • Modeling clay
  • Petri dishes, plastic
  • 0.5‐ml thin‐walled microcentrifuge tubes, sterile
  • Gloves sprayed with antistatic spray
  • Bright‐field microscope mounted on a vibration isolation table and equipped with high‐resolution micromanipulator (Narashige) and rotating stage
  • Thermal cycler with heated lid
  • BioSpin P6 columns (Bio‐Rad)
  • Additional reagents and equipment for mammalian cell culture ( appendix 3G), preparation of metaphase spreads from cultured cells (unit 4.1), chromosome banding (unit 4.2), agarose gel electrophoresis (unit 2.7), and ethanol precipitation of DNA ( appendix 3C)
NOTE: Exceptional care must be taken in preparing reagents for chromosome microdissection. Each working solution is prepared, divided into single‐use aliquots, tested, and used for microdissection only if satisfactory. When preparing solutions, wear gloves and, to the extent it is possible, conduct all manipulations in a laminar flow hood.

Basic Protocol 2: Isolating Region‐Specific cDNAs by Microdissection‐Mediated cDNA Capture

  Materials
  • Cultured cells
  • 0.5 mg/ml linkered cDNA (e.g., unit 6.3, CPMB UNIT )
  • 1.0 mg/ml C 0t 1 DNA
  • 1.0 mg/ml herring sperm DNA
  • 3 M sodium acetate, pH 5.2 ( appendix 2D)
  • 100% ethanol, ice‐cold
  • 70% ethanol
  • recipeMMcC hybridization solution (see recipe)
  • SSC ( appendix 2D)/0.1% (v/v) Tween 20, 65°C
  • 0.5× SSC/0.1% (v/v) Tween 20, 65°C
  • 0.1× SSC/0.1% (v/v) Tween 20, 65°C
  • SSC, room temperature
  • recipeMMcC PCR mix (see recipe)
  • Low‐molecular‐weight DNA size markers
  • 100 µM primer for UDG cloning, e.g., CUA Catch A primer: 5′‐CUACUACUACUAGAGTAGAATTCTAATATCTC‐3′
  • Glass slides
  • 22 × 60–mm glass coverslips
  • Rubber cement
  • Moist chamber (Fig. )
  • 45°, 65°, and 66°C water baths
  • Coplin jars
  • Bright‐field microscope mounted on a vibration isolation table and equipped with high‐resolution micromanipulator (Narashige) and rotating stage
  • Microdissection needles (see protocol 1, steps and )
  • 0.2‐ml thin‐walled microcentrifuge tubes
  • Thermal cycler with a heated lid
  • Additional reagents and equipment for preparing metaphase chromosomes (unit 4.1), precipitating DNA ( appendix 3C), in situ hybridization (unit 4.3), Giemsa banding (unit 4.2), microdissecting chromosomes (see protocol 1), PCR (unit 6.3), agarose gel electrophoresis (unit 2.7), purifying PCR products using spin‐column chromatography ( appendix 3E), quantifying PCR products by spectrophotometry ( appendix 3D), and UDG cloning (unit 6.1).
NOTE: Exceptional care must be taken when preparing reagents for chromosome microdissection. Each working solution is prepared, divided into single‐use aliquots, tested, and accepted for microdissection only if satisfactory. When preparing solutions, wear gloves and, to the extent it is possible, conduct all manipulations in a laminar flow hood.
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Figures

Videos

Literature Cited

Literature Cited
   Bohlander, S.K., Espinosa, R., Le Beau, M., Rowley, J.D., and Diaz, M.O. 1992. A method for the rapid sequence‐independent amplification of microdissected chromosomal material. Genomics 13:1322‐1324.
   Elkahloun, A.G., Meltzer, P.S., Guan, X.Y., McNinch, J.S., Trent, J.M., and de Jong, P.J. 1996. Isolation of a cosmid sublibrary for a region of chromosome 12 frequently amplified in human cancers using a complex chromosome microdissection probe. Genomics 31:343‐347.
   Fisher, E.M., Cavanna, J.S., and Brown, S.D. 1985. Microdissection and microcloning of the mouse X chromosome. Proc. Natl. Acad. Sci. U.S.A. 82:5846‐5249.
   Gracia, E., Fischer, U., Elkahloun, A.G., Trent, J.M., Meese, E., and Meltzer, P.S. 1996. Isolation of genes amplified in human cancers by microdissection‐mediated cDNA capture. Hum. Mol. Genet. 5:595‐600.
   Gracia, E., Ray, M., Polymeropoulos, M., Dehijiaa, A., Meltzer, P.S., and Trent, J.M. 1997. Isolation of chromosome‐specific ESTs by microdissection‐mediated cDNA capture. Genome Res. 7:100‐107.
   Guan, X.Y., Meltzer, P.S., Cao, J., and Trent, J.M. 1992. Rapid generation of region‐specific genomic clones by chromosome microdissection: Isolation of DNA from a region frequently deleted in malignant melanoma. Genomics 14:680‐684.
   Guan, X.Y., Trent, J.M., and Meltzer, P.S. 1993. Generation of band‐specific painting probes from a single microdissected chromosome. Hum. Mol. Genet. 2:1117‐1121.
   Guan, X.Y., Meltzer, P.S., and Trent, J.M. 1994a. Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection. Genomics 22:101‐107.
   Guan, X.Y., Meltzer, P.S., Dalton, W.S., and Trent, J.M. 1994b. Identification of cryptic sites of DNA sequence amplification in human breast cancer by chromosome microdissection. Nature Genet. 8:155‐161.
   Guan, X.Y., Zhang, H., Bittner, M.L., Jiang, Y., Meltzer, P.S., and Trent, J.M. 1996a. Chromosome arm painting probes. Nature Genet. 12:10‐11.
   Guan, X.Y., Xu, J., Anzick, S.L., Zhang, H., Trent, J.M., and Meltzer, P.S. 1996b. Selection of transcribed sequences from microdissected DNA: Isolation of genes within an amplified region at 20q11‐q13.2 in breast cancer. Cancer Res. 56:3446‐3450.
   Johnson, D.H. 1990. Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence‐independent amplification of DNA. Genomics 6:243‐251.
   Kinzler, K.W. and Vogelstein, B. 1989. Whole‐genome PCR: Application to the identification of sequences bound by gene regulatory proteins. Nucl. Acids Res. 7:3645‐3653.
   Ludecke, H.‐J., Senger, G., Claussen, U., and Horsthemke, B. 1989. Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338:348‐350.
   Meltzer, P.S., Guan, X.Y., Burgess, A., and Trent, J.M. 1992. Rapid generation of region‐specific probes by chromosome microdissection and their application. Nature Genet. 1:24‐28.
   Meltzer, P.S., Guan, X.Y., and Trent, J.M. 1993. Telomere capture stabilizes chromosome breakage. Nature Genet. 4:252‐255.
   Meltzer, P.S., Guan, X.Y., Su, Y.A., Gracia, E., and Trent, J.M. 1997. Identification of region‐specific genes by chromosome microdissection. Cancer Genet. Cytogenet. 93:29‐32.
   Scalenghe, F., Turco, K.E., Edstrom, J.‐E., Pirrotta, V., and Melli, M. 1981. Microdissection and cloning of DNA from a specific region of Drosophia melanogaster polytene chromosomes. Chromosoma 82:205‐216.
   Schrock, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson‐Smith, M.A., Ning, Y., Ledbetter, D.H., Bar‐Am, I., Soenksen, D., Garini, Y., and Ried, T. 1996. Multicolor spectral karyotyping of human chromosomes. Science 273:494‐497.
   Speicher, M.R., Gwyn‐Ballard, S., and Ward, D.C. 1996. Karyotyping human chromosomes by combinatorial multifluor FISH. Nature Genet. 12:368‐375.
   Su, Y.A., Guan, X.Y., Trent, J.M., and Meltzer, P.S. 1994. Direct isolation of expressed sequences encoded within a homogeneously staining region by chromosome microdissection. Proc. Natl. Acad. Sci. U.S.A. 91:9121‐9125.
   Telenius, H., Carter, N.P., Bebb, C.E., Nordenskjold, M., Ponder, B.A., and Tunnacliffe, A. 1992. Degenerate oligonucleotide–primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13:718‐725.
   Zhang, J., Meltzer, P.S., Jenkins, R., Guan, X.Y., and Trent, J. 1993a. Application of chromosome microdissection probes for elucidation of BCR‐ABL fusion and variant Philadelphia chromosome translocations in chronic myelogenous leukemia. Blood 81:3365‐3371.
   Zhang, J., Trent, J.M., and Meltzer, P.S. 1993b. Rapid isolation and characterization of amplified DNA by chromosome microdissection: Identification of IGF1R amplification in malignant melanoma. Oncogene 8:2827‐2831.
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