Single‐Strand Conformation Polymorphism Analysis Using Capillary Electrophoresis

Lars Allan Larsen1, Michael Christiansen1, Jens Vuust1, Paal Skytt Andersen1

1 Statens Serum Institut, Copenhagen
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.12
DOI:  10.1002/0471142905.hg0712s36
Online Posting Date:  May, 2003
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Abstract

Single‐strand conformation polymorphism (SSCP) is one of the most frequently used mutation detection methods. This unit describes a method of SSCP with automated analysis by capillary electrophoresis in order to increase the capacity and throughput. A protocol is provided for sample preparation. For a medium throughput laboratory, a single capillary instrument, as described in this unit, may be quite sufficient. In many cases, however, screening for mutations in large population groups requires a high throughput, and this is best obtained through the use of a multi‐capillary instrument, as discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Sample Preparation for Capillary Electrophoresis
  • Basic Protocol 2: Automated Capillary Electrophoresis Using an ABI 310 Genetic Analyzer
  • Basic Protocol 3: Automated Capillary Array Electrophoresis Using an ABI PRISM 3100 Genetic Analyzer
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Sample Preparation for Capillary Electrophoresis

  Materials
  • 5′ FAM‐labeled forward primer (e.g., MWG‐Biotech)
  • 5′ HEX‐labeled reverse primer (e.g., MWG‐Biotech)
  • dNTPs (10 mM each; appendix 2D)
  • 10× PCR buffer (Roche Diagnostics)
  • Pwo DNA polymerase (Roche Diagnostics)
  • 20 to 60 ng/µl genomic DNA
  • Milli‐Q water
  • 25 mM MgSO 4
  • 2% (w/v) agarose gel
  • 7% (v/v) GeneScan polymer (Applied Biosystems)
  • 10× genetic analyzer buffer with EDTA (Applied Biosystems)
  • 87% (v/v) glycerol solution (Merck)
  • Deionized formamide
  • 0.3 N NaOH
  • DNA size standard (e.g., GeneScan 500 ROX, Applied Biosystems)
  • Software for T m calculation (e.g., AnnHyb, http://annhyb.free.fr/; or Oligo, Molecular Biology Insights; optional)
  • 0.2‐ml PCR tubes
  • Thermal cycler (e.g., PTC200 DNA engine, MJ Research)
  • 50‐ml polypropylene tubes
  • ABI PRISM genetic analyzer (Applied Biosystems)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Basic Protocol 2: Automated Capillary Electrophoresis Using an ABI 310 Genetic Analyzer

  Materials
  • PCR products (see protocol 1)
  • 5% nondenaturing polymer, with 5% glycerol added (see protocol 1, step )
  • 10× genetic analyzer buffer with EDTA (Applied Biosystems)
  • Milli‐Q purified water
  • ABI PRISM 310 genetic analyzer (Applied Biosystems)
  • Genetic analyzer capillaries, 50‐µm i.d. (Applied Biosystems)
  • GeneScan analysis software (Applied Biosystems)
  • Genotyper software (optional, Applied Biosystems)
  • Additional reagents and equipment for DNA sequencing (unit 7.7)

Basic Protocol 3: Automated Capillary Array Electrophoresis Using an ABI PRISM 3100 Genetic Analyzer

  Materials
  • PCR products (see protocol 1)
  • 5% nondenaturing polymer, with 5% glycerol added (see protocol 1, step )
  • 10× genetic analyzer buffer with EDTA (Applied Biosystems)
  • Milli‐Q purified water
  • ABI PRISM 3100 genetic analyzer with 16 capillaries (Applied Biosystems)
  • 36‐cm GeneScan capillary array (50‐µm i.d.; Applied Biosystems)
  • GeneScan NT version 3.7 (Applied Biosystems)
  • Genotyper NT version 3.7 (optional, Applied Biosystems)
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Figures

Videos

Literature Cited

Literature Cited
   Inazuka, M., Tahira, T., and Hayashi, K. 1996. One‐tube post‐PCR fluorescent labeling of DNA fragments. Genome Res. 6:551‐557.
   Inazuka, M., Wenz, H.M., Sakabe, M., Tahira, T., and Hayashi, K. 1997. A streamlined mutation detection system: Multicolor post‐PCR fluorescence labeling and single‐strand conformational polymorphism analysis by capillary electrophoresis. Genome Res. 7:1094‐1103.
   Iwahana, H., Fujimura, M., Takahashi, Y., Iwabuchi, T., Yoshimoto, K., and Itakura, M. 1996. Multiple fluorescence‐based PCR‐SSCP analysis using internal fluorescent labeling of PCR products. Biotechniques 21:510‐519.
   Kuypers, A.W., Willems, P.M., van der Schans, M.J., Linssen, P.C., Wessels, H.M., de Bruijn, C.H., Everaerts, F.M., and Mensink, E.J. 1993. Detection of point mutations in DNA using capillary electrophoresis in a polymer network. J. Chromatogr. 621:149‐156.
   Larsen, L.A., Christiansen, M., Vuust, J., and Andersen, P.S. 1999. High‐throughput single‐strand conformation polymorphism analysis by automated capillary electrophoresis: Robust multiplex analysis and pattern‐based identification of allelic variants. Hum. Mutat. 13:318‐327.
   Ren, J. 2000. High‐throughput single‐strand conformation polymorphism analysis by capillary electrophoresis. J. Chromatogr. B. Biomed. Sci. Appl. 741:115‐128.
   Sasaki, T., Tahira, T., Suzuki, A., Higasa, K., Kukita, Y., Baba, S., and Hayashi, K. 2001. Precise estimation of allele frequencies of single‐nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA. Am. J. Hum. Genet. 68:214‐218.
   Sheffield, V.C., Beck, J.S., Kwitek, A.E., Sandstrom, D.W., and Stone, E.M. 1993. The sensitivity of single‐strand conformation polymorphism analysis for the detection of single base substitutions. Genomics 16:325‐332.
   Walz, T., Geisel, J., Bodis, M., Knapp, J.P., and Herrmann, W. 2000. Fluorescence‐based single‐strand conformation polymorphism analysis of mutations by capillary electrophoresis. Electrophoresis 21:375‐379.
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