Characterization of (CA)n Microsatellite Repeats from Large‐Insert Clones

Mike Litt1, David Browne1

1 Oregon Health Sciences University, Portland, Oregon
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.4
DOI:  10.1002/0471142905.hg0204s00
Online Posting Date:  May, 2001
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Abstract

The most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit determination of sequences flanking the microsatellites. When cosmids or large‐insert phage clones are used as primary sources of (CA)n repeat markers, they have traditionally been subcloned into plasmid vectors such as pUC18 or M13 mp18/19 cloning vectors to obtain fragments of suitable size for DNA sequencing. This unit presents an alternative approach whereby a set of degenerate sequencing primers that anneal directly to (CA)n microsatellites can be used to determine sequences that are inaccessible with vector‐derived primers. Because the primers anneal to the repeat and not to the vector, they can be used with subclones containing inserts of several kilobases and should, in theory, always give sequence in the regions directly flanking the repeat. Degeneracy at the 3 end of each of these primers prevents elongation of primers that have annealed out‐of‐register.The most laborious part of developing (CA)n microsatellite repeats as genetic markers is constructing DNA clones to permit.

     
 
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Table of Contents

  • Basic Protocol 1: Sequencing of (CA)nRepeats Using Degenerate Primers
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Sequencing of (CA)nRepeats Using Degenerate Primers

  Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Cosmid or phage clone containing insert DNA with (CA) n repeats
  • Restriction endonucleases with 4‐ and 6‐base recognition sites and appropriate buffers
  • recipeHybridization buffer, prewarmed to 65°C (see recipe)
  • recipe100 pmol/µl (CA) 15 oligonucleotide probe (see recipe)
  • 10 mCi/ml [γ‐32P]ATP (3000 mCi/mmol)
  • Low‐stringency wash solution: 6× SSC/0.1% (w/v) SDS, prewarmed to 65°C
  • High‐stringency wash solution: 0.2× SSC/0.1% (w/v) SDS, prewarmed to 65°C
  • recipe100 pmol/µl (CA) 11(A/G/T) and (GT) 11(A/C/T) degenerate sequencing primers (see recipe)
  • AmpliTaq Cycle sequencing system (Perkin‐Elmer Cetus) or equivalent reagents (CPMB UNIT )
  • Thermal cycler (Perkin‐Elmer Cetus GeneAmp PCR System 9600 or equivalent)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), Southern blotting (unit 2.7), 5′‐end labeling of oligonucleotides ( appendix 3E), measuring radioactivity by TCA precipitation ( appendix 3E), subcloning (CPMB UNIT ), preparation of phage DNA or plasmid minipreps for dideoxy sequencing (CPMB UNIT ), and colony hybridization (unit 2.3)CAUTION: [γ‐32P]ATP is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
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Figures

Videos

Literature Cited

   Browne, D.L. and Litt, M. 1992. Characterization of (CA)n microsatellites with degenerate sequencing primers. Nucl. Acids Res. 20:141.
   Craxton, M. 1991. Linear amplification sequencing, a powerful method for sequencing DNA. Methods 3:1.
   Church, G.M. and Kieffer‐Higgins, S. 1988. Multiplex DNA sequencing. Science 240:185‐188.
Key Reference
   Browne, D.L. and Litt, M. 1992. See above
  Original description of this methodology.
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