Generation of FISH probes using laser microbeam microdissection and application to clinical molecular cytogenetics.
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-->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.
Shim
Sung Han
SH
Department of Medical Genetics, College of Medicine, Hanyang University, Seoul 133-791, Korea.
Kyhm
Jee Hong
JH
Chung
Sung Ro
SR
Kim
Seung Ryong
SR
Park
Moon Il
MI
Lee
Chul-Hoon
CH
Cho
Youl-Hee
YH
D020223:Chromosome Painting / Q000379:methods; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D020732:Cytogenetic Analysis; D005819:Genetic Markers; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007834:Lasers; D008677:Metaphase / Q000235:genetics; D042282:Microdissection / Q000379:methods; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction
D005819:Genetic Markers