Abstract
Chimerism, the presence of noncontiguous DNA fragments in the same clone, is one of the most common problems encountered when working with yeast artificial chromosomes (YACs). Its frequency can vary among the different libraries, but on average, 40–60% of YAC clones among the most used libraries are chimeric (1). To determine if a given YAC clone is chimeric, the most obvious approach is to isolate both ends of the insert and map them to assess if they have the same chromosomal origin, However, this approach could be time consuming, especially when an investigator deals with many YACs isolated from the same genomic locus and wants to identify very quickly the clones to be characterized. An alternative approach involves the fluorescent in situ hybridization (FISH) of Alu polymerase chain reaction (Alu-PCR) products, from YAC DNA to human metaphase chromosomes. This method, although very powerful, has the disadvantages of requiring expertise in preparing metaphase chromosomal spreads, and the need for specialized equipment.
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
Green, E. D., Riethman, H. C., Dutchik, J. E., and Olson, M. V. (1991) Detection and characterization of chimeric yeast artificial-chromosome clones. Genomics 11, 658–669.
Ledbetter, S. A., Garcia-Heras, J., and Ledbetter, D. H. (1990) “PCR-karyotype” of human chromosomes in somatic cell hybrids. Genomics 8, 614–622.
Breukel, C., Wijnen, J., Tops, C., Klift, H., Dauwerse, H., and Khan, P. M. (1990) Vector-Alu PCR: a rapid step in mapping cosmids and YACs. Nucleic Acids Res. 18, 3097.
Feinberg, A. P. and Vogelstein, B. ( 1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266,267.
Zoghbi, H. Y. and Chinault, A. C. (1993) Generation of YAC contigs by walking, in YACs: A User Guide (Nelson, D. L. and Brownstein, B. H., eds.), Freeman, New York, pp. 93–112.
Banfi, S., Ledbetter, S. A., Chinault, A. C., and Zoghbi, H. Y. (1992) An easy and rapid method for the detection of chimeric yeast artificial chromosomes clones. Nucleic Acids Res. 20, 1814.
Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F., Ramirez-Sobs, R., Webster, T. D., et al. (1989) Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources. Proc. Natl. Acad. Sci. USA 88, 6157–6161.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc.
About this protocol
Cite this protocol
Banfi, S., Zoghbi, H.Y. (1996). Detection of Chimerism in YAC Clones. In: Markie, D. (eds) YAC Protocols. Methods in Molecular Biology™, vol 54. Humana Press. https://doi.org/10.1385/0-89603-313-9:115
Download citation
DOI: https://doi.org/10.1385/0-89603-313-9:115
Publisher Name: Humana Press
Print ISBN: 978-0-89603-313-9
Online ISBN: 978-1-59259-541-9
eBook Packages: Springer Protocols