Restriction Fragment Length Polymorphism Analysis

John Jarcho1

1 Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.7
DOI:  10.1002/0471142905.hg0207s01
Online Posting Date:  May, 2001
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Abstract

This unit describes Restriction Fragment Length Polymorphism (RFLP) analysis, which utilizes restriction endonuclease digestion to identify DNA sequence polymorphisms in genes or DNA regions of interest. When investigating families for inheritance of an RFLP, aliquots of genomic DNA from individual family members are digested to completion with the restriction enzyme known to generate the polymorphism of interest. After size fractionation on an agarose gel, DNA is transferred to a membrane by capillary action in a high‐salt buffer. The gel is first treated with NaOH to denature DNA, and after neutralization, the gel is placed between buffer‐soaked filter paper and a sheet of membrane. Labeled probe is hybridized overnight to the Southern blot. The blot is washed under conditions designed to remove all nonspecifically adherent probe and exposed to X‐ray film. Identified fragment sizes differ among individuals and can be traced from generation to generation.

     
 
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Table of Contents

  • Basic Protocol 1: RFLP Analysis by Southern Blot Hybridization
  • Support Protocol 1: Agarose Gel Electrophoresis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: RFLP Analysis by Southern Blot Hybridization

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • 0.5 to 2.0 µg/µl sample DNA ( appendix 3B) in H 2O or TE buffer
  • Restriction endonuclease and 10× buffer
  • 50 mM spermidine
  • 10× gel loading buffer ( appendix 2D)
  • Agarose gel ( protocol 2)
  • recipeDenaturing solution (see recipe)
  • recipeNeutralizing solution (see recipe)
  • 20× and 2× SSC ( appendix 2D)
  • recipeHybridization cocktail (see recipe), 42°C
  • recipe2 mg/ml herring sperm DNA (see recipe)
  • 108 to 109 cpm/µg labeled probe DNA ( appendix 3E)
  • recipe20 mg/ml human blocking DNA (see recipe)
  • 2× SSC/0.1% (w/v) SDS ( appendix 2D)
  • 0.2× SSC/0.1% (w/v) SDS ( appendix 2D), room temperature and 65°C
  • recipeStripping solution (see recipe), 90°C
  • 65°, 90°, and 100°C water baths
  • Glass or plastic box or dish, preferably ≥3‐liter volume
Two rectangular plastic or glass plates, one 20 cm wide and long enough to bridge the sides of the box or dish (Fig. ) and one 20 × 20 cm
  • Whatman 3MM filter paper, 20‐cm‐wide roll and sheets
  • Nylon or nitrocellulose membrane, 20 × 20 cm
  • Glass hybridization tube or plastic hybridization bag
  • 42°C hybridization oven with platform shaker or rotator
CAUTION: Radiolabeled probe DNA and hybridization cocktail are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: If glass hybridization tubes and hybridization oven are used, they should be obtained from the same manufacturer (e.g., the Techne Hybridization Oven, VWR Scientific or Fisher).

Support Protocol 1: Agarose Gel Electrophoresis

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Agarose, electrophoresis gradeElectrophoresis buffer: TAE bufferor TBE buffer ( appendix 2D)
  • 10 mg/ml ethidium bromide ( appendix 2D)
  • Sample DNA
  • 10× gel loading buffer ( appendix 2D)
  • DNA molecular size markers: e.g., λ phage cut with HindIII
  • Gel casting platform
  • Gel comb
  • Horizontal gel electrophoresis apparatus
  • DC power supply
  • Polaroid MP4 camera with orange filter (Kodak Wratten no. 23A), UV‐blocking filter (Kodak Wratten no. 2B), and film cassettes
CAUTION: Ethidium bromide is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
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Figures

Videos

Literature Cited

   Barker, D., Schafer, M., and White, R. 1984. Restriction sites containing CpG show a higher frequency of polymorphism in human DNA. Cell 36:131‐138.
   Botstein, D., White, R.L., Skolnick, M., and Davis, R.W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32:314‐331.
   Gusella, J.F., Wexler, N.S., Conneally, P.M., Naylor, S.L., Anderson, M.A., Tanzi, R.E., Watkins, P.C., Ottina, K., Wallace, M.R., Sakaguchi, A.Y., Young, A.B., Shoulson, I., Bonilla, E., and Martin, J.B. 1983. A polymorphic DNA marker genetically linked to Huntington's disease. Nature 306:234‐238.
   NIH/CEPH Collaborative Mapping Group. 1992a. A comprehensive genetic linkage map of the human genome. Science 258:67‐86.
   NIH/CEPH Collaborative Mapping Group. 1992b. A comprehensive genetic linkage map of the human genome. Appendix. Science 258:148‐162.
   White, R. and Lalouel, J.‐M. 1987. Investigation of genetic linkage in human families. In Advances in Human Genetics, Vol. 16 (H. Harris and K. Hirschhorn, eds.) pp.121‐228. Plenum, New York.
   Wijsman, E.M. 1984. Optimizing selection of restriction enzymes in the search for DNA variants Nucl. Acids Res. 12:9209‐9226.
Key Reference
   Botstein, D., et al., 1980. See above.
  This reference was the first to describe the principle of linkage analysis using RFLPs. It also contains a thorough discussion of theoretical considerations.
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