Overview of Strategies for Screening YAC Libraries and Analyzing YAC Clones

David D. Chaplin1, Bernard H. Brownstein1

1 Howard Hughes Medical Institute and Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 6.9
DOI:  10.1002/0471142727.mb0609s20
Online Posting Date:  May, 2001
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Abstract

This unit provides an introduction to the use of yeast artificial chromosome‐bearing yeast clones (hereafter referred to as YAC clones) in genome analysis. It describes criteria for designing a polymerase chain reaction (PCR) assay to be used in screening a YAC core library and discusses the rationale for verification and characterization of YAC clones obtained from these core laboratories. Protocols for maintaining YAC clones, analyzing YAC insert structure, preparing YAC DNA, and subcloning YAC inserts into other vectors are presented elsewhere in this volume.

     
 
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Table of Contents

  • Generating YAC Libraries
  • YAC Library Screening by a Core Laboratory
  • Designing a Locus‐Specific PCR Assay for Screening
  • Analyzing Individual YAC Clones
  • Construction and Analysis of a YAC‐Insert Subligrary
  • Figures
     
 
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Materials

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Figures

Videos

Literature Cited

Literature Cited
   Abidi, F.E., Wada, M., Little, R.D., and Schlessinger, D. 1990. Yeast artificial chromosomes containing human Xq24‐Xq28 DNA: Library construction and representation of probe sequences. Genomics 7:363‐376.
   Bronson, S.K., Pei, J., Taillon‐Miller, P., Chorney, M.J., Geraghty, D.E., and Chaplin, D.D. 1991. Isolation and characterization of yeast artificial chromosome clones linking the HLA‐B and HLA‐C loci. Proc. Natl. Acad. Sci. U.S.A. 88:1671‐1675.
   Burke, D.T., Carle, G.F., and Olson, M.V. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806‐812.
   Burke, D.T., Rossi, J.M., Leung, J., Koos, D.S., and Tilghman, S.M. 1991. A mouse genomic library of yeast artificial chromosome clones. Mamm. Genome 1:65‐69.
   Green, E.D. and Olson, M.V. 1990. Systematic screening of yeast artificial chromosome libraries by use of the polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 87:1213‐1217.
   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.
   Green, E.D. 1992. Physical mapping of human chromosomes: Generation of chromosome‐specific sequence‐tagged sites (STS). Methods Mol. Genet. In press.
   Hillier, L. and Green, P. 1991. A computer program for choosing PCR and DNA sequencing primers. PCR Meth. Appl. 1:124‐128.
   Olson, M., Hood, L., Cantor, C., and Botstein, D. 1989. A common language for physical mapping of the human genome. Science 245:1434‐1435.
   Proffitt, J.H., Davie, J.R., Swinton, D., and Hattman, S. 1984. 5‐Methylcytosine is not detectable in Saccharomyces cerevisiae DNA. Mol. Cell Biol. 4:985‐988.
   Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., Powell, S., Anand, R., Smith, J.C., and Markham, A.F. 1990. A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucl. Acids Res. 18:2887‐2890.
   Rossi, J.M., Burke, D.T., Leung, J.C., Koos, D.S., Chen, H., and Tilghman, S.M. 1992. Genome analysis using a yeast artificial chromosome library with mouse DNA inserts. Proc. Natl. Acad. Sci. U.S.A. 89:2456‐2460.
   Vollrath, D. and Davis, R.W. 1987. Resolution of DNA molecules greater than 5 megabases by contour‐clamped homogeneous electric fields. Nucl. Acids Res. 15:7865‐7876.
Key Reference
   Burke et al., 1987. See above.
  Initial description of the YAC cloning system, covering general features of library construction.
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