FISH with whole-chromosome or region-specific painting probes made from either flow-sorted or microdissected chromosomes has revolutionized cytogenetics. The generation of paints from flow-sorted chromosomes relies on the use of an expensive and sophisticated fluorescence-activated cell sorter and suspensions of freshly prepared chromosomes. The preparation of paints from microdissected materials requires an inverted microscope with appropriate micromanipulators and metaphase chromosome spreads on coverslips. Painting probes made from flow-sorted chromosomes generally have better chromosomal coverage and can be used in a wide range of applications between unrelated species, while region-specific probes from microdissection enable higher resolution analyses restricted to comparative painting between closely related species. Here we provide detailed protocols on the generation of probes from both flow-sorted and microdissected chromosomes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Backx L, Van Esch H, Melotte C, Kosyakova N, Starke H, Frijns JP, Liehr T, Vermeesch JR (2007) Array painting using microdissected chromosomes to map chromosomal breakpoints. Cytogenet Genome Res 116:158–166
Carter NP, Ferguson-Smith MA, Perryman MT, Telenius H, Pelmear AH, Leversha MA, Glancy MT, Wood SL, Cook K, Dyson HM (1992) Reverse chromosome painting: a method for the rapid analysis of aberrant chromosomes in clinical cytogenetics. J Med Genet 29:299–307
Guan XY, Meltzer PS, Cao J, Trent JM (1992) Rapid generation of region-specific genomic clones by chromosome microdissection: isolation of DNA from a region frequently deleted in malig nant melanoma. Genomics 14:680–684
Jauch A, Wienberg J, Stanyon R, Arnold N, Tofanelli S, Ishida T, Cremer T (1992) Reconstruction of genomic rearrangements in great apes and gibbons by chromosome painting. Proc Natl Acad Sci USA 89:8611–8615
Liehr T, Heller A, Starke H, Rubtsov N, Trifonov V, Mrasek K, Weise A, Kuechler A, Claussen U (2002) Microdissection based high resolution multicolor banding for all 24 human chromo somes. Int J Mol Med 9:335–339
Liehr T, Starke H, Heller A, Kosyakova N, Mrasek K, Gross M, Karst C, Steinhaeuser U, Hunstig F, Fickelscher I, Kuechler A, Trifonov V, Romanenko SA, Weise A (2006) Multicolor fluores cence in situ hybridization (FISH) applied to FISH-banding. Cytogenet Genome Res 114: 240–244
Meltzer PS, Guan X Y, Burgess A, Trent JM (1992) Rapid generation of region specific probes by chromosome microdissection and their application. Nat Genet 1:24–28
Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J (1988) Fluorescence in situ hybridization with human chromosome-specific libraries — detection of trisomy-21 and translocations of chromosome-4. Proc Natl Acad Sci USA 85:9138–9142
Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high-sensivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934–2938
Rabbitts P, Impey H, Heppell-Parton A, Langford C, Tease C, Lowe N, Bailey D, Ferguson-Smith MA, Carter NP (1995) Chromosome specific paints from a high resolution flow karyotype of the mouse. Nat Genet 9:369–375
Scalenghe F, Turco E, Edstrom JE, Pirrotta V, Melli M (1981) Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes. Chromosoma 82:205–216
Scherthan H, Cremer T, Arnason U, Weier HU, Lima-de-Faria M, Frönicke L (1994) Comparative chromosome painting discloses homologous segments in distantly related mammals. Nat Genet 6:342–347
Senger G, Lüdecke HJ, Horsthemke B, Claussen U (1990) Microdissection of banded human chromosomes. Hum Genet 84:507–511
Starke H, Raida M, Trifonov V, Clement JH, Loncarevic IF, Heller A, Bleck C, Nietzel A, Rubtsov N, Claussen U, Liehr T (2001) Molecular cytogenetic characterization of an acquired super numerary minute marker chromosome as sole abnormality in a case clinically diagnosed as Philadelphia negative CML. Br J Haematol 113(2):435–438
Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BA, Tunnacliffe A (1992a) Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13:718–725
Telenius H, Pelmear AH, Tunnacliffe A, Carter NP, Behmel A, Ferguson-Smith MA, Nordenskjold M, Pfragner R, Ponder BA (1992b) Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4:257–263
Trifonov VA, Perelman PL, Kawada S-I, Iwasa MA, Oda S-I, Graphodatsky AS (2002) Complex structure of B-chromosomes in two mammalian species: Apodemus peninsulae (Rodentia) and Nyctereutes procyonoides (Carnivora) revealed by microdissection. Chromosome Res 10(2): 109–116
Trifonov V, Seidel J, Starke H, Prechtel M, Beensen V, Ziegler M, Hartmann I, Heller A, Nietzel A, Claussen U, Liehr T (2003) Enlarged chromosome 13 p-arm hiding a cryptic partial trisomy 6p22.2-pter. Prenat Diagn 23(5):427–430
Trifonov V, Karst C, Claussen U, Mrasek K, Michel S, Avner P, Liehr T (2005) Microdissection-derived murine Mcb probes from somatic cell hybrids. J Histochem Cytochem 53:791–792
Weimer J, Kiechle M, Senger G, Wiedemann U, Ovens-Raeder A, Schuierer S, Kautza M, Siebert R, Arnold N (1999) An easy and reliable procedure of microdissection technique for the analysis of chromosomal breakpoints and marker chromosomes. Chromosome Res 7:355–362
Wienberg J, Jauch A, Stanyon R, Cremer T (1990) Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization. Genomics 8:347–350
Yang F, Carter NP, Shi L, Ferguson-Smith MA (1995) A comparative study of karyotypes of muntjacs by chromosome painting. Chromosoma 103:642–652
Yang F, O'Brien P, Milne B, Graphodatsky A, Solansky N, Trifonov V, Rens W, Sargan D, Ferguson-Smith M (1999) A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps. Genomics 62:189–202
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Springer-Verlag Berlin Heidelberg
About this protocol
Cite this protocol
Yang, F., Trifonov, V., Ng, B.L., Kosyakova, N., Carter, N.P. (2009). Generation of Paint Probes by Flow-Sorted and Microdissected Chromosomes. In: Liehr, T. (eds) Fluorescence In Situ Hybridization (FISH) — Application Guide. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-70581-9_3
Download citation
DOI: https://doi.org/10.1007/978-3-540-70581-9_3
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-70580-2
Online ISBN: 978-3-540-70581-9
eBook Packages: Springer Protocols