Besides the well-known applications of bacterial artificial chromosomes (BACs) in classical molecular genetics, BACs are also used for molecular cytogenetic studies. BACs, as well as other locus-specific probes like cDNA, plasmids, cosmids, fosmids, P1-clones or yeast artificial chromosomes (YACs), can be labeled with fluorochromes and applied in FISH experiments. Various applications are possible, like gene mapping, FISH banding, determination of chromosomal breakpoints, characterization of derivative chromosomes, studies of interphase architecture, and karyotypic evolution studies. Here the basic principle of hybridizing BACs in situ on chromosome preparations is outlined. Moreover, an overview of possible issues that can be studied using BACs as FISH probes is provided. Finally, a shortened and more efficient FISH protocol using microwave treatment which yields results that can be evaluated within a few hours is presented.
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
Birren BW, Tachi-iri Y, Kim UJ, Nguyen M, Shizuya H, Korenberg JR, Simon MI (1996)A human chromosome 22 fosmid resource: mapping and analysis of 96 clones. Genomics 34:97–106
Gisselsson D, Palsson E, Hoglund M, Domanski H, Mertens F, Pandis N, Sciot R, Dal Cin P, Bridge JA, Mandahl N (2002) Differentially amplified chromosome 12 sequences in low- and high-grade osteosarcoma. Genes Chromosomes Cancer 33:133–140
Liehr T (2006) Application of YACs in fluorescence in situ hybridization (FISH). In: Alesandair M (ed) Methods in molecular biology YAC protocols, 2nd edn. Humana, Totowa, NJ, pp. 175–186, ISBN 1–58829–612–1
Liehr T, Weise A, Heller A, Starke H, Mrasek K, Kuechler A, Weier HU, Claussen U (2002)Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries. Cytogenet Genome Res 97:43–50
Liehr T, Starke H, Senger G, Melotte C, Weise A, Vermeesch JR (2006) Overrepresentation of small supernumerary marker chromosomes (sSMC) from chromosome 6 origin in cases with multiple sSMC. Am J Med Genet A 140:46–51
Mark H, Wyandt H, Huang X, Milunsky J (2005) Delineation of a supernumerary marker chromosome utilizing a multimodal approach of G-banding, fluorescent in situ hybridization, confirmatory P1 artificial chromosome fluorescent in situ hybridization, and high-resolution comparative genomic hybridization. Clin Genet 68:146–151
Michels-Rautenstrauss KG, Mardin C Y, Budde WM, Liehr T, Polansky J, Nguyen T, Timmerman V, Van Broeckhoven C, Naumann GO, Pfeiffer RA, Rautenstrauss BW (1998) Juvenile open angle glaucoma: fine mapping of the TIGR gene to 1q24.3-q25.2 and mutation analysis. Hum Genet 102:103–106
Nogami M, Nogami O, Kagotani K, Okumura M, Taguchi H, Ikemura T, Okumura K (2000)Intranuclear arrangement of human chromosome 12 correlates to large-scale replication domains. Chromosoma 108:514–522
Pietrzak J, Mrasek K, Obersztyn E, Stankiewicz P, Kosyakova N, Weise A, Cheung SW, Cai WW, von Eggeling F, Mazurczak T, Bocian E, Liehr T (2007) Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4,8, 18, and 21 in three patients. J Appl Genet 48:167–175
Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934–2938
Rubtsov N, Senger G, Kuzcera H, Neumann A, Kelbova C, Junker K, Beensen V, Claussen U (1996) Interstitial deletion of chromosome 6q: precise definition of the breakpoints by micro-dissection, DNA amplification, and reverse painting. Hum Genet 97:705–709
Schmidt S, Claussen U, Liehr T, Weise A (2005) Evolution versus constitution: differences in chromosomal inversion. Hum Genet 117:213–219
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Tsuchiya D, Matsumoto A, Covert SF, Bronson CR, Taga M (2002) Physical mapping of plasmid and cosmid clones in filamentous fungi by fiber-FISH. Fungal Genet Biol 37:22–28
von Deimling F, Scharf JM, Liehr T, Rothe M, Kelter AR, Albers P, Dietrich WF, Kunkel LM, Wernert N, Wirth B (1999) Human and mouse RAD17 genes: Identification, localization, genomic structure and histological expression pattern in normal testis and seminoma. Hum Genet 105:17–27
Weise A, Harbarth P, Claussen U, Liehr T (2003) Fluorescence in situ hybridization (FISH) on human chromosomes using photoprobe biotin-labeled probes. J Histochem Cytochem 51:549–551
Weise A, Starke H, Mrasek K, Claussen U, Liehr T (2005a) New insights into the evolution of chromosome 1. Cytogenet Genome Res 108:217–222
Weise A, Liehr T, Claussen U, Halbhuber K-J (2005b) Increased efficiency of fluorescence in situ hybridization (FISH) using the microwave. J Histochem Cytochem 53:1301–1303
Weise A, Gross M, Mrasek K, Mkrtchyan H, Horsthemke B, Jonsrud C, von Eggeling F, Hinreiner S, Witthuhn V, Claussen U, Liehr T (2008) Parental-origin-determination FISH (pod-FISH) distinguishes homologous human chromosomes on a single cell level. Int J Mol Med 21:189–200
Acknowledgments
Acknowledgments Supported in part by the Evangelische Studienwerk e.V. Villigst, IZKF Jena (Start-up S16, TP 3.7), Friedrich Schiller University Jena, TMWFK (B307–04004), Stiftung Leukämie and Stefan-Morsch-Stiftung.
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
Weise, A. et al. (2009). ISH Probes Derived from BACs, Including Microwave Treatment for Better FISH Results. 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_4
Download citation
DOI: https://doi.org/10.1007/978-3-540-70581-9_4
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-70580-2
Online ISBN: 978-3-540-70581-9
eBook Packages: Springer Protocols