Preparation of DNA from Fixed, Paraffin‐Embedded Tissue

Edward A. Fox1

1 Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3I
DOI:  10.1002/0471142905.hga03is08
Online Posting Date:  May, 2001
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Abstract

Purification of DNA from fixed, paraffin‐embedded tissue (PET) requires the removal of paraffin, proteins, and, in some cases, heavy metal ions. The resulting DNA is not generally suitable for Southern blot analysis. However, the polymerase chain reaction (PCR) can be used to amplify short regions of this DNA. The Basic Protocol is rapid and involves only a small number of manipulations, thus minimizing the possibility of contamination. The Alternate Protocol involves additional steps including extractions with xylenes and phenol/chloroform. This procedure is recommended as the primary approach when tissue has not been fixed in an optimal manner or as a secondary approach when the Basic Protocol has failed to produce satisfactory DNA.

     
 
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Table of Contents

  • Basic Protocol 1: DNA Isolation Using Mixed‐Bed Chelating Resin
  • Alternate Protocol 1: DNA Isolation from Nonoptimally Fixed Tissue
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: DNA Isolation Using Mixed‐Bed Chelating Resin

  Materials
  • Paraffin‐embedded tissue sections, ∼5 to 10 µm thick
  • recipePET‐B solution (see recipe)
  • recipeProteinase K stock solution (see recipe)
  • recipeChelex 100 slurry (see recipe)
  • 65°C and boiling water baths
  • Additional reagents and equipment for quantitation of DNA ( appendix 3D)

Alternate Protocol 1: DNA Isolation from Nonoptimally Fixed Tissue

  • Xylenes
  • 100% and 70% ethanol
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • 24:1 (v/v) chloroform/isoamyl alcohol ( appendix 3C)
  • 3 M sodium acetate, pH 5.2 ( appendix 2D; filter sterilize with 0.45‐µm filter and store up to 6 months at room temperature)
  • TE buffer, pH 8.0 ( appendix 2D)
  • Additional reagents and equipment for phenol/chloroform extraction ( appendix 3C) and DNA quantitation ( appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
   Campell, J. and Gerard, G. 1994. Proteinase K stability. Focus (BRL) 16(1):22‐23.
   Chaw, Y., Crane, L., Lange, P., and Shapiro, R. 1980. Isolation and identification of cross‐links from formaldehyde‐treated nucleic acids. Biochemistry 19:5525‐5531.
   Feldman, M. 1973. Reactions of nucleic acids and nucleoproteins with formaldehyde. Prog. Nucleic Acid Res. Mol. Biol. 13:1‐49.
   Greer, C., Peterson, S., Kiviat, N., and Manos, M. 1991. PCR amplification from paraffin‐embedded tissues. Anat. Pathol. 95:117‐124.
   Jackson, V. 1978. Studies on histone organization in the nucleosome using formaldehyde as a reversible cross‐linking agent. Cell 15:945‐954.
   Jackson, D., Lewis, F., Taylor, G., Boylston, A., and Quirke, P. 1990. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. J. Clin. Pathol. 43:499‐504.
   Karlsen, R., Kalantari, M., Chitemerere, M., Johansson, B., and Hagmar, B. 1994. Modifications of human and viral deoxyribonucleic acid by formaldehyde fixation. Lab. Invest. 71:604‐611.
Key Reference
   Mies, C. 1994. Molecular biological analysis of paraffin‐embedded tissues. Hum. Pathol. 25:555‐560.
  An overview of technical and medical considerations in using nucleic acids extracted from fixed, paraffin‐embedded tissues.
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