Colony Hybridization to Screen for Microsatellites

Thomas J. Hudson1

1 Whitehead Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.3
DOI:  10.1002/0471142905.hg0203s14
Online Posting Date:  May, 2001
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Abstract

Simple sequence length polymorphisms (SSLPs) for use as genetic markers are derived from the microsatellite repeat sequences found abundantly throughout eukaryotic genomes. Microsatellite DNA is identified in libraries of cloned DNA by colony hybridization to oligonucleotide probes containing simple sequence repeat (SSR) DNA, a common procedure in molecular biology laboratories. Plasmid or phagemid colonies are transferred to a nylon filter, grown briefly, fixed, and hybridized with one or more 32Pā€labeled oligonucleotide probes. This protocol is adapted for the rapid processing of many filters at once and optimized for identification of long stretches of uninterrupted repetitive DNA sequences.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Small‐insert plasmid or phagemid library (unit 2.1 or unit 2.2)
  • 150‐mm LB plates (for plasmid library) or 150‐mm 2× TY plates made with top agarose (for phagemid library), containing appropriate selective antibiotic (see appendix 2D for plate, top agarose, and antibiotic recipes)
  • recipeChurch's hybridization buffer (see recipe)
  • 32P‐labeled synthetic oligonucleotide probe ( appendix 3E)
  • Washing solution: 1× SSC/0.1% (w/v) SDS
  • LB medium ( appendix 2D; for phagemid library)
  • 137‐mm nylon colony/plaque transfer membrane discs
  • Blunt‐ended filter forceps
  • Whatman 3MM filter paper
  • Round, sealable plastic hybridization containers, 20‐cm diameter
  • Water bath set at selected hybridization temperature
  • UV‐transparent plastic wrap (e.g., Saran Wrap)
CAUTION:32P is hazardous; see appendix 2A for guidelines on handling, storage and disposal.
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Figures

Videos

Literature Cited

   Church, G.M. and Gilbert, W. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. U.S.A. 81:1991‐1995.
   Hudson, T.J., Engelstein, M., Lee, M.K., Ho, E.C., Rubenfield, M.J., Adams, C.P., Housman, D.E., and Dracopoli, N.C. 1992. Isolation and chromosomal assignment of 100 highly informative human simple sequence repeat polymorphisms. Genomics 13:622‐629.
   Weber, J.L. 1990. Informativeness of human (dC‐dA)n⋅(dG‐dT)n polymorphisms. Genomics 7:524‐530.
Key Reference
   Hudson et al., 1992. See above
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