Human Genetics

, Volume 84, Issue 6, pp 507–511 | Cite as

Microdissection of banded human chromosomes

  • Gabriele Senger
  • Hermann-Josef Lüdecke
  • Bernhard Horsthemke
  • Uwe Claussen
Original Investigations

Summary

Physical dissection of metaphase chromosomes is the most straightforward approach for the isolation of DNA sequences from specific chromosome regions. However, conventional microdissection techniques are too crude and inefficient for analysis of the human genome. Here we describe a technique for the precise dissection of single bands from GTG-banded chromosomes. Cells from normal amniotic fluid cell cultures are harvested by the pipette method. Microdissection is performed on an inverted microscope (magnification 1250 x) with the help of extended siliconized glass needles and an electronically controlled micromanipulator. Enzymatic amplification of the dissected DNA allows the construction of band-specific DNA libraries from as few as 20 dissected chromosome fragments.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bates GP, Wainwright BJ, Williamson R, Brown SDM (1986) Microdissection and microcloning from the short arm of human chromosome 2. Mol Cell Biol 6:3826–3830Google Scholar
  2. Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314–331PubMedGoogle Scholar
  3. Buiting K, Neumann M, Lüdecke H-J, Senger G, Claussen U, Antich J, Passarge E, Horsthemke B (1989) Microdissection of potential gene sequences from the Prader-Willi syndrome chromosome region. Genomics (in press)Google Scholar
  4. Claussen U (1980) The pipette method: a new rapid technique for chromosome analysis in prenatal diagnosis. Hum Genet 54:277–278Google Scholar
  5. Claussen U, Klein R, Schmidt M (1986) A pipette method for rapid karyotyping in prenatal diagnosis. Prenat Diagn 6:401–408Google Scholar
  6. Edström J-E, Kaiser R, Röhme D (1987) Microcloning of mammalian metaphase chromosomes. Methods Enzymol 151:503–516Google Scholar
  7. Fisher EMC, Cavanna JS, Brown SDM (1985) Microdissection and microcloning of the mouse X-chromosome. Proc Natl Acad Sci USA 82:5846–5849Google Scholar
  8. Greenfield AJ, Brown SDM (1987) Microdissection and microcloning from the proximal region of mouse chromosome 7: isolation of clones genetically linked to the Pudgy locus. Genomics 1:153–158Google Scholar
  9. Kaiser R, Weber J, Grzeschik K-H, Edström J-E, Driesel A, Zengerling S, Buchwald M, Tsui LC, Olek K (1987) Microdissection and microcloning of the long arm of human chromosome 7. Mol Biol Rep 12:3–6Google Scholar
  10. Lichter P, Cremer T, Tang C-JC, Watkins PC, Manuelidis L, Ward DC (1988) Rapid detection of human chromosome 21 aberrations by in situ hybridisation. Proc Natl Acad Sci USA 85:9664–9668Google Scholar
  11. Lüdecke H-J, Senger G, Claussen U, Horsthemke B (1989) Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338:348–350Google Scholar
  12. Lüdecke H-J, Senger G, Claussen U, Horsthemke B (1990) Construction and characterization of band-specific DNA libraries. Hum Genet 84:512–516Google Scholar
  13. McKusick V (1988) Mendelian inheritance in man, 8th edn. Johns Hopkins University Press, Baltimore LondonGoogle Scholar
  14. Mezzanotte R, Vanni R, Flore O, Ferrucci L, Sumner AT (1988) Ageing of fixed cytological preparations produces degradation of chromosomal DNA. Cytogenet Cell Genet 48:60–62Google Scholar
  15. Richa J, Lo CW (1989) Introduction of human DNA into mouse eggs by injection of dissection chromosome fragments. Science 245:175–177Google Scholar
  16. Röhme D, Fox H, Hermann B, Frischauf A-M, Edström J-E, Mains P, Silver LM, Lehrach H (1984) Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes. Cell 36:783–788Google Scholar
  17. Scalenghe F, Turco E, Edström J-E, Pirrotta V, Melli M (1981) Microdissection and cloning of DNA from specific region of Drosophilia melanogaster polytene chromosomes. Chromosoma 82:205–216Google Scholar
  18. Weith A, Winking H, Brackmann B, Boldyreff B, Traut W (1987) Microclones from a mouse germ line HSR detect amplification and complex rearrangements of DNA sequences. EMBO J 6:1295–1300Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Gabriele Senger
    • 1
  • Hermann-Josef Lüdecke
    • 2
  • Bernhard Horsthemke
    • 2
  • Uwe Claussen
    • 1
  1. 1.Institut für Humangenetik der UniversitätErlangenGermany
  2. 2.Institut für Humangenetik, UniversitätsklinikumEssen 1Germany

Personalised recommendations