Automated Fluorescent Genotyping

Jeff Hall1, Elizabeth Nanthakumar1

1 Sequana Therapeutics, Inc., La Jolla, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.8
DOI:  10.1002/0471142905.hg0208s14
Online Posting Date:  May, 2001
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Abstract

With the publication of a comprehensive human genetic map consisting of over 5000 microsatellite markers, reagents are in hand to undertake large‐scale genotyping projects. This unit describes the basic methodology, optimization of markers, and allele calling.With the publication of a comprehensive human genetic map consisting of over 5000 microsatellite markers, reagents are in hand.

     
 
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Table of Contents

  • Basic Protocol 1: PCR Amplification of SSLPs for Automated Fluorescent Genotyping
  • Support Protocol 1: Pooling Fluorescently Labeled PCR Products for Genotype Analysis
  • Support Protocol 2: Gel Electrophoresis of Pooled PCR Products
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: PCR Amplification of SSLPs for Automated Fluorescent Genotyping

  Materials
  • Patient DNA samples at a concentration of 4 ng/µl in TE buffer
  • 10× PCR buffer (Perkin‐Elmer)
  • 10 mM 4dNTP mix ( appendix 2D or Perkin‐Elmer)
  • 25 mM MgCl 2
  • 20 µM fluorochrome‐labeled forward primer for each microsatellite marker
  • 20 µM reverse primer for each microsatellite marker
  • 5 U/µl AmpliTaq Gold DNA polymerase (Perkin‐Elmer)
  • 96‐well plates (Robbins)
  • Drying oven
  • Multichannel pipettor
  • 96‐well strip caps (Robbins) or 96‐well plastic plate‐sealing film (Costar)
  • 5‐ml and 50‐ml tubes
  • Reagent trays, sterile
  • Thermal cycler(s) equipped to handle 96‐well plates

Support Protocol 1: Pooling Fluorescently Labeled PCR Products for Genotype Analysis

  Materials
  • Fluorochrome‐labeled PCR products (see protocol 1)
  • 4× BB loading dye: 0.167% (w/v) bromphenol blue/30% (v/v) glycerol
  • 50 µg/ml DNA size standard (e.g., DNA Mass Ladder, Life Technologies)
  • Genescan 500 Tamra size standard (Perkin‐Elmer)
  • Beckman CS‐6 centrifuge and Beckman TS‐2000 rotor (or equivalent) with 96‐well plate adaptors
  • 96‐well plates
  • Multichannel pipettor
  • 96‐well plastic plate‐sealing film (Costar)
  • 90°C oven
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Support Protocol 2: Gel Electrophoresis of Pooled PCR Products

  Materials
  • Gel‐loading plate (see protocol 2)
  • recipe6% (w/v), 24‐cm well‐to‐read acrylamide gel (see recipe)
  • 1× TBE electrophoresis buffer ( appendix 2D)
  • Automated fluorescent sequencer (e.g., ABI 373 or 377 for four‐color detection, Perkin‐Elmer; or Pharmacia, LICOR for single‐color detection) with computer and mouse
  • Genescan 672 Collection, Analysis, and Genotyper software (Perkin‐Elmer)
  • 60‐cc syringe with needles
  • Lane‐indicator strip, plastic (Perkin‐Elmer; optional)
  • Micropipettor
  • Gel‐loading micropipettor tips
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Figures

Videos

Literature Cited

Literature Cited
   Beckmann, J.S., Tomfohrde, J., Barnes, R.I., Williams, M., Broux, O., Richard, I., Weissenbach, J., and Bowcock, A.M. 1993. A linkage map of human chromosome 15 with an average resolution of 2 cM and containing 55 polymorphic microsatellites. Hum. Mol. Genet. 2:2019‐2030.
   Brownstein, M.J., Carpten, J.D., and Smith, J.R. 1996. Modulation of non‐templated nucleotide addition by Taq polymerase: Primer modifications that facilitate genotyping. BioTechniques 20:1004‐1010
   Diehl, S.R., Ziegle, J., Buck, G.A., Reynolds, T.R., and Weber, J.L. 1990. Automated genotyping of human DNA polymorphisms. Am. J. Hum. Genet. 47:A177.
   Litt, M. and Luty, J.A. 1989. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44:397‐401.
   Smeets, H.J.M., Brunner, H.G., Ropers, H.H., and Wieringa, B. 1989. Use of variable simple sequence motifs as genetic markers: Application to study of myotonic dystrophy. Hum. Genet. 83:245‐251.
   Weber, J.L. and May, P.E. 1989. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Gen. 44:338‐396.
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