Screening Large‐Insert Libraries by PCR

A.Craig Chinault1, Nat L. Sternberg2

1 Baylor College of Medicine, Houston, Texas, 2 Du Pont/Merck Pharmaceuticals, Glenolden, Pennsylvania
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.5
DOI:  10.1002/0471142905.hg0505s00
Online Posting Date:  May, 2001
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Abstract

This unit describes procedures for screening large‐insert libraries by multistep polymerase chain reaction (PCR) analysis of DNA samples from clone pools. In the basic protocol, PCR amplification and agarose gel electrophoresis are used to identify successively smaller pools of DNA or colonies that contain clones with the appropriate‐sized amplification product. In the last screening step, individual clones are identified. The first and second support protocols describe the preparation of DNA and yeast‐cell pools for screening, and the first alternate protocol describes the preparation of crude lysates suitable for PCR from individual clones or small‐scale pools.

     
 
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Table of Contents

  • Basic Protocol 1: Screening YAC Pool DNA by PCR
  • Support Protocol 1: Preparation of Primary and Secondary Pool DNA
  • Support Protocol 2: Preparation of Row/Column Clone Pools
  • Alternate Protocol 1: Preparation of Crude Lysates
  • Alternate Protocol 2: Organizing and Screening P1 Libraries
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Screening YAC Pool DNA by PCR

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D)
  • 2.5 mM 4dNTP mix ( appendix 2D)
  • Oligonucleotide primers 1 and 2 (forward and reverse), 100 µM each (store at −20°C)
  • 5 U/µl Taq or Amplitaq DNA polymerase (Perkin‐Elmer Cetus)
  • 10 ng/µl primary pool DNA sample (first support protocol)
  • 10 ng/µl total human genomic DNA ( appendix 3B)
  • Mineral oil
  • DNA molecular size standards
  • 10 ng/µl secondary pool DNA sample (first support protocol)
  • Yeast cells from row and column pools (second support protocol)
  • 150‐mm recipeAHC plates (see recipe)
  • 1.5‐ and 0.5‐ml microcentrifuge tubes
  • 30°C incubator
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Support Protocol 1: Preparation of Primary and Secondary Pool DNA

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • YAC library stocks in 96‐well microtiter plates (unit 5.2)
  • 150‐mm recipeAHC plates (see recipe)
  • recipeCell isolation solution (see recipe)
  • TE buffer, pH 8.0
  • recipeSpheroplast suspension solution (make fresh; see recipe)
  • recipeSpheroplast lysis solution (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • Isopropanol
  • 500 µg/ml DNase‐free RNase (Boehringer Mannheim or appendix 2D; store at −20°C)
  • 132‐mm nylon membrane filters (Oncor or Amersham)
  • 96‐pin replicator (Washington University Instrument Machine Shop, V & P Scientific, or Thomas Scientific) or robotic gridding workstation with replicating tool (e.g., Biomek 1000, Beckman)
  • Bent glass rods, sterile
  • 50‐ml centrifuge tubes
  • IEC Centra‐8R centrifuge with 216 swinging‐bucket rotor and adaptors (or equivalent)
  • Additional reagents and equipment for quantitation of DNA ( appendix 3D)
CAUTION: Phenol and chloroform are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 2: Preparation of Row/Column Clone Pools

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • YAC library stocks in 96‐well microtiter plates (unit 5.2)
  • recipeAHC plates (see recipe)
  • recipeYPD medium (see recipe)
  • 80% (v/v) glycerol
  • 132‐mm nylon membrane filter (Oncor or Amersham)
  • 96‐pin replicator (Washington University Instrument Machine Shop) or robotic gridding workstation with replicating tool (e.g., Biomek 1000,Beckman)
  • Glass plate

Alternate Protocol 1: Preparation of Crude Lysates

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • recipeLysate buffer (see recipe)
  • 15 mg/ml Lyticase (Sigma; store at −20°C)
  • 100°C water bath
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Figures

Videos

Literature Cited

   Amemiya, C.T., Alegria‐Hartman, M.J., Aslanidis, C., Chen, C., Nilolic, J., Gingrich, J.C., and de Jong, P.J. 1992. A two‐dimensional YAC pooling strategy for library screening via STS and Alu‐PCR methods. Nucl. Acids Res. 20:2559‐2563.
   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 chromosome clones. Nucl. Acids Res. 20:1814.
   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.
   Chaplin, D.D. and Brownstein, B.H. 1992. Overview of strategies for screening YAC libraries and analyzing YAC clones. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl, eds.) pp. 6.9.1‐6.9.7. Greene Publishing Associates and John Wiley & Sons, New York.
   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., Riethan, H.C., Dutchik, J.E., and Olson, M.V. 1991. Detection and characterization of chimeric yeast artificial‐chromosome clones. Genomics 11:658‐669.
   Kwiatkowski, T.J., Zoghbi, H.Y., Ledbetter, S.A., Ellison, K.A., and Chinault, A.C. 1990. Rapid identification of yeast artificial chromosome clones by matrix pooling and crude lysate PCR. Nucl. Acids Res. 18:7191‐7192.
   Nagata, S., Taira, H., Hall, A., Johnsrud, L., Streuli, M., Escodi, J., Boll, W., Cantell, K., and Weissmann, C. 1980. Synthesis in E. coli of a polypeptide with human leukocyte interferon activity. Nature 284:316‐320.
   Olson, M., Hood, L., Cantor, C., and Botstein, D. 1989. A common language for physical mapping of the human genome. Science 245:1434‐1435.
   Pierce, J., Saver, B., and Sternberg, N. 1992. A positive selection vector for cloning HMW DNA by the bacteriophage P1 system: improved cloning efficacy. Proc. Natl. Acad. Sci. U.S.A. 89:2056‐2060.
   Rossi, J.M., Burke, D.T., Leung, J.C.M., Koos, D.S., Chen, H., and Tilghman, S.M. 1992. Genomic analysis using a yeast artificial chromosome library with mouse DNA inserts. Proc. Natl. Acad. Sci. U.S.A. 89:2456‐2460.
   Shizuya, H., Birren, B., Kim, U.‐J., Mancino, V., Slepak, T., Yoshiaki, T., and Simon, M. 1992. Cloning and stable maintenance of 300‐kilobase pair fragments of human DNA in Escherichia coli using an F‐factor based vector. Proc. Natl. Acad. Sci. U.S.A. 89:8794‐8797.
Key Reference
   Green and Olson, 1990. See above.
  This paper introduced the use of multistep PCR for identification of specific YAC clones.
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