Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Generation of FISH Probes Using Laser Microbeam Microdissection and Application to Clinical Molecular Cytogenetics

  • Shim, Sung-Han (Department of Medical Genetics, College of Medicine, College of Medicine, Hanyang University) ;
  • Kyhm, Jee-Hong (Department of Medical Genetics, College of Medicine, College of Medicine, Hanyang University) ;
  • Chung, Sung-Ro (Department of Obstetrics and Gynecology, College of Medicine, Hanyang University) ;
  • Kim, Seung-Ryong (Department of Obstetrics and Gynecology, College of Medicine, Hanyang University) ;
  • Park, Moon-Il (Department of Obstetrics and Gynecology, College of Medicine, Hanyang University) ;
  • Lee, Chul-Hoon (Department of Medical Genetics, College of Medicine, College of Medicine, Hanyang University) ;
  • Cho, Youl-Hee (Department of Medical Genetics, College of Medicine, College of Medicine, Hanyang University)
  • Published : 2007.07.31
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Abstract

Chromosome microdissection and the reverse FISH technique is one of the most useful methods for the identification of structurally abnormal chromosomes. In particular, the laser microbeam microdissection (LMM) method allows rapid isolation of a target chromosome or a specific region of chromosomes without damage of genetic materials and contamination. Isolated chromosomes were directly amplified by the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), and then the FISH probes labeled with spectrum green- or spectrum red-dUTP were generated by nick-translation. Whole chromosome painting (WCP) probes were successfully generated from only 5 copies of the chromosome. With this method, we produced 24 WCP probes for each human chromosome. We also tried to characterize a marker chromosome, which seemed to be originated from chromosome 11 on conventional banding technique. The marker chromosomes were isolated by the LMM method and analyzed by reverse FISH. We elucidated that the marker chromosome was originated from the short arm of chromosome 5 ($5p11{\to}pter$). A fully automated and computer-controlled LMM method is a very simple laboratory procedure, and enables rapid and precise characterization of various chromosome abnormalities.

Keywords

References

  1. Christian, A. T., H. E. Garcia, and J. D. Tucker. 1999. PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosome. Mamm. Genome 10: 628-631 https://doi.org/10.1007/s003359901058
  2. Deng, H. X., K. I. Yoshiura, R. W. Dirks, N. Harada, T. Hirota, K. Tsukamoto, Y. Jinno, and N. Nikawa. 1992. Chromosome-band-specific painting: Chromosome in situ suppression hybridization using PCR products from microdissected chromosome band as a probe pool. Hum. Genet. 89: 13-17 https://doi.org/10.1007/BF00207034
  3. Engelen, M. J. J., J. C. Albrechts, G. J. H. Hamers, and J. P. M. Geraedts. 1998. A simple and efficient method for microdissection and microFISH. J. Med. Genet. 35: 265-268 https://doi.org/10.1136/jmg.35.4.265
  4. Goldammer, T., R. Weikard, R. M. Brunner, and M. Schwerin. 1996. Generation of chromosome fragment specific bovine DNA sequences by microdissection and DOP-PCR. Mamm. Genome 7: 291-296 https://doi.org/10.1007/s003359900085
  5. Guan, X. Y, M. T. Jeffrey, and P. S. Meltzer. 1993. Generation of band-specific painting probes from single microdissected chromosome. Hum. Mol. Genet. 2: 1117-1121 https://doi.org/10.1093/hmg/2.8.1117
  6. Guan, X. Y., P. S. Meltzer, and J. M. Trent. 1994. Rapid generation of whole chromosome painting probes (WCP) by chromosome microdissection. Genomics 22: 101-107 https://doi.org/10.1006/geno.1994.1350
  7. Hozier, J. C., B. K. Hall, K. R. Sims, M. C. Liechty, L. ChenLui, and L. M. Davis. 1996. Chromosome microdissectionbased techniques for genome analysis. Methods 9: 74-83 https://doi.org/10.1006/meth.1996.0010
  8. Lengauer, C., H. Riethman, and T. Cremer. 1990. Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers. Hum. Genet. 86: 1-6
  9. Lichter, P., T. Cremer, J. Borden, L. Manuelidis, and D. C. Ward. 1998. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet. 80: 224-234
  10. Meltzer, P. S., X. Y. Guan, A. Burgess, and J. M. Trent. 1992. Rapid generation of region specific probes by chromosome microdissection and their application. Nature Genet. 1: 24-28 https://doi.org/10.1038/ng0492-24
  11. Muller-Navia, J. and A. N. E. Schleiermacher. 1995. Complete and precise characterization of marker chromosomes by application of microdissection in prenatal diagnosis. Hum. Genet. 96: 661-667 https://doi.org/10.1007/BF00210295
  12. Pinkel, D., T. Straume, and J. W. Gary. 1986. Cytogenetic analysis using qualitative, high sensitivity fluorescence hybridization. Proc. Natl. Acad. Sci. USA 82: 2934-2938
  13. Schermelleh, L., S. Thalhammer, W. Heckl, H. Posl, T. Cremer, K. Schutze, and M. Cremer. 1999. Laser microdissection and laser pressure catapulting for the generation of chromosomespecific paint probes. Biotechniques 27: 362-267
  14. Telenius, H., N. P. Carter, C. E. Bebb, M. Nordenskjold, B. A. J. Ponder, and A. Tunnacliffe. 1992. Degenerated oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13: 718-725 https://doi.org/10.1016/0888-7543(92)90147-K
  15. Verma, R. S. and A. Babu. 1995. Human Chromosomes: Principles and Techniques, 2nd Ed. McGraw-Hill Inc., New York
  16. Vierbach, R., G. Schwannitz, and M. M. Nothen. 1994. Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes. Hum. Genet. 93: 663-667
  17. Xiao, Y., F. Darroudi, A. G. J. Kuipers, J. H. D. Jong, P. D. Boer, and A. T. Natarajan. 1996. Generation of mouse chromosome painting probes by DOP-PCR amplification of microdissected meiotic chromosome. Cytogenet. Cell Genet. 75: 63-66 https://doi.org/10.1159/000134458
  18. Xu, J., C. T. Fong, E. Cedrone, J. Sullivan, and N. Wang. 1998. Prenatal identification of de novo marker chromosomes using micro-FISH approach. Clin. Genet. 53: 490-496 https://doi.org/10.1111/j.1399-0004.1998.tb02602.x