Generating Subclones from Large‐Insert Genomic Clones

Anthony P. Monaco1, Zoia Larin2

1 John Radcliffe Hospital, Headington, Oxford, 2 University of Oxford, South Parks Road, Oxford
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.11
DOI:  10.1002/0471142905.hg0511s03
Online Posting Date:  May, 2001
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Abstract

This unit provides a simple protocol for generating a partial‐digest sublibrary of yeast DNA containing a YAC of interest. The starting material is high‐molecular‐weight chromosomal DNA embedded in agarose plugs. Many genome equivalents of these YAC subclones in bacteriophage or cosmid vectors can be plated and screened by hybridization with total human DNA to identify clones that originate from the human portion of the YAC. The human‐positive clones can be picked into 96‐well microtiter plates for spotting on membranes. Once stored in ordered arrays, the YAC subclones can be constructed into contigs using an end‐probe or Alu‐PCR hybridization strategy, or a gel fingerprinting technique.This unit provides a simple protocol for generating a partial‐digest sublibrary of yeast DNA containing a YAC of interest.

     
 
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Table of Contents

  • Basic Protocol 1: Construction of Bacteriophage and Cosmid Libraries from Whole YAC DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Construction of Bacteriophage and Cosmid Libraries from Whole YAC DNA

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Yeast culture containing YAC of interest
  • TE buffer, pH 7.5 ( appendix 2D), 50°C and room temperature
  • recipe10× T4 DNA polymerase salts (see recipe)
  • 1 mg/ml bovine serum albumin (BSA)
  • 5 mM dithiothreitol (DTT)
  • MboI and BamHI restriction endonucleases
  • 0.3% (w/v) agarose gel
  • λ DNA digested with HindIII and λ DNA digested with SalI
  • 1 U/µl β‐agarase I (New England Biolabs)
  • 1 U/µl calf intestine alkaline phosphatase (CIP; Boehringer Mannheim)
  • recipeT4 DNA polymerase buffer (prepare fresh; see recipe)
  • 0.15 M nitrilotriacetic acid (NTA, or trinitriloacetic acid; e.g., from BDH), adjusted to pH 8.0 with NaOH
  • 10 mg/ml Dextran T‐40 (Pharmacia Biotech)
  • Phenol equilibrated ( appendix 3B) with 100 mM Tris⋅Cl, pH 8.0 ( appendix 2D)
  • Chloroform
  • 4 M NaCl
  • 70% ethanol
  • 10× and 1× T4 DNA ligase buffer ( appendix 2D; omit BSA)
  • 400 U/µl T4 DNA ligase (cohesive end units; New England Biolabs)
  • 10 U/µl T4 polynucleotide kinase (New England Biolabs)
  • Lambda (Lambda EMBL3 or Lambda DASHII) or SuperCos 1 vector kit with appropriate E. coli host strain (all from Stratagene)
  • Gigapack II Gold (for lambda vector) or Gigapack II XL (for cosmid vector) packaging extract (both from Stratagene)
  • 37° and 68°C heating blocks
  • 16°C water bath
  • Additional reagents and equipment for preparation of yeast chromosomes in agarose blocks using LiDS (unit 5.1), agarose gel electrophoresis (unit 2.7), phenol extraction and ethanol precipitation of DNA ( appendix 3C), and plating, screening, and purification of clones from bacteriophage or cosmid genomic libraries (CPMB units 6.1 6.6)
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Figures

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Literature Cited

   Baxendale, S., Bates, G.P., MacDonald, M.E., Gusella, J.F., and Lehrach, H. 1991. The direct screening of cosmid libraries with YAC clones. Nucl. Acids Res 19:6651.
   Cohen, D., Chumakov, I., and Weissenbach, J. 1993. A first‐generation physical map of the human genome. Nature 366:698‐701.
   Coulson, A.R., Sulston, J., Brenner, S., and Karn, J. 1986. Towards a physical map of the genome of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A 83:7821‐7825.
   Coulson, A., Kozono, Y., Lutterbach, B., Shownkeen, R., Sulston, J., and Waterston, R. 1991. YACs and the C. elegans genome. BioEssays 13:413‐417.
   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.
   Larin, Z., Monaco, A.P., and Lehrach, H. 1991. Yeast artificial chromosome libraries containing large inserts from mouse and human DNA. Proc. Natl. Acad. Sci. U.S.A 88:4123‐4127.
   Pieretti, M., Tonlorenzi, R., and Ballabio, A. 1991. Rapid assembly of lambda phage contigs within YAC clones. Nucl. Acids Res 19:2795‐2796.
Key Reference
   Berger, S.L. and Kimmel, A.R. (eds). 1987. Guide to Molecular Cloning Techniques. VI. Genomic Cloning. Methods Enzymol. 152:173‐215.
  Contains several chapters on different topics related to construction of partial‐digest genomic libraries in bacteriophage and cosmid vectors.
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