DNA Isolation from Mammalian Samples

Ximeng Liu1, Shuko Harada1

1 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.14
DOI:  10.1002/0471142727.mb0214s102
Online Posting Date:  April, 2013
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Abstract

DNA isolation is a fundamental initial step for molecular genetic studies. Two quite different methodologies are described in this unit: silica spin column and phenol/chloroform extraction. Currently, the most commonly used technique is the silica‐based spin column, although phenol/chloroform extraction is still widely used. This unit also presents basic procedures for extraction of DNA from both fresh tissues and formalin‐fixed, paraffin‐embedded (FFPE) tissue samples. Curr. Protoc. Mol. Biol. 102:2.14.1–2.14.13. © 2013 by John Wiley & Sons, Inc.

Keywords: DNA isolation; silica spin column; FFPE (formalin‐fixed paraffin‐embedded) tissue; automated extraction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: DNA Extraction from Blood or Bone Marrow Using Silica‐Gel Column
  • Basic Protocol 2: DNA Extraction from Blood or Bone Marrow Using Phenol/Chloroform
  • Basic Protocol 3: DNA Extraction from Formalin‐Fixed Paraffin‐Embedded (FFPE) Tissue
  • Alternate Protocol 1: Zymo Pinpoint Dissection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: DNA Extraction from Blood or Bone Marrow Using Silica‐Gel Column

  Materials
  • Lysis buffer (see recipe)
  • Specimen (one of the types described below)
    • Whole blood (1 to 3 ml) or bone marrow (1 ml) collected in EDTA (lavender‐top tube), stored at 2° to 8°C for no more than 2 days before extraction
    • Cultured cells (monolayer or suspension)
    • Fresh or frozen tissue
    • Formalin‐fixed paraffin‐embedded (FFPE) samples ( protocol 3 and protocol 4)
  • 20 mg/ml proteinase K (e.g., Qiagen, Life Technologies, New England Biolabs)
  • 100% ethanol
  • Silica binding mix (see recipe) or commercially available spin columns with silica‐gel‐membrane (e.g., Qiagen QIAmp DNA Mini or Blood Mini Kit)
  • Wash buffer 1 (from spin column kit or see recipe)
  • Wash buffer 2 (from spin column kit or see recipe)
  • TE, pH 8.0 ( appendix 22)
  • Fluorescent dye for DNA quantitation, e.g., PicoGreen, Molecular Probes (optional)
  • Water bath, 56°C
  • Microcentrifuge, with rotor capable of 20,000 × g (14,000 rpm)
  • 2‐ml microcentrifuge tubes, if using spin columns that reguire 2‐ml tubes
  • 1.5‐ or 2‐ml microcentrifuge tubes for final elution (DNase‐ and RNase‐free)
  • UV spectrophotometer or fluorometer for DNA quantitation (optional)

Basic Protocol 2: DNA Extraction from Blood or Bone Marrow Using Phenol/Chloroform

  Materials
  • Specimen (one of the types described below)
    • Whole blood (1 to 3 ml) or bone marrow (1 ml) collected in EDTA (lavender‐top tube), stored at 2° to 8°C for no more than 2 days before extraction
    • Cultured cells (monolayer or suspension)
    • Fresh or frozen tissue
    • Formalin‐fixed paraffin‐embedded (FFPE) samples ( protocol 3 and protocol 4)
  • Extraction buffer (see recipe)
  • 20 mg/ml proteinase K (e.g., Qiagen, Life Technologies, New England Biolabs)
  • Phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) (see recipe)
  • Isopropanol
  • 70% (v/v) ethanol, prepared from reagent‐grade 100% ethanol
  • TE, pH 8.0 ( appendix 22; optional)
  • Fluorescent dye for DNA quantitation, e.g., PicoGreen, Molecular Probes (optional)
  • 56° to 60°C water bath
  • Sterile, RNase‐free, fine‐tip transfer pipets
  • UV spectrophotometer or fluorometer for DNA quantitation (optional)

Basic Protocol 3: DNA Extraction from Formalin‐Fixed Paraffin‐Embedded (FFPE) Tissue

  Materials
  • Paraffin block containing embedded tissue, or unstained sections on slides
  • 10% bleach (for cleaning microtome)
  • Xylene (Fisher #X5‐500) [store at room temperature (15° to 25°C) in a fireproof cabinet]
  • 96% to 100% ethanol
  • Lysis buffer (see recipe)
  • Microtome and microtome blade
  • Uncharged microscope slides
  • Slide‐marking pen, e.g., Pilot extra fine point permanent marker
  • 1.5‐ml microcentrifuge tubes, DNase and RNase free (Fisher #05‐408‐129)
  • Chemical hood
  • Sterile glass disposable serological pipets or adjustable pipet and sterile filter tips
  • Sterile, fine tip disposable transfer pipets
  • Additional reagents and equipment for preparing paraffin sections (unit 14.1), staining with hematoxylin and eosin (unit 14.5), and DNA isolation (Basic Protocol protocol 11 or protocol 22)

Alternate Protocol 1: Zymo Pinpoint Dissection

  Materials
  • FFPE sections on “plus” slides (typically 2 to 5 slides are used depending on the size of the tissue of interest) with H&E‐stained slide adjacent to the sections
  • Xylene
  • 100% and 95% (v/v) ethanol
  • Pinpoint Solution (Zymo #D3001‐1)
  • Extraction Buffer (Zymo #D3001‐3)
  • Proteinase K and storage buffer (Zymo #D3001‐2; prior to use, add 1040 µl proteinase K storage buffer to the proteinase K)
  • Coplin jar or plastic slide mailer
  • PCR tubes
  • Thermocycler (ABI 9600/9700) or heating block
  • Zymo Spin I Columns or QIAmp DNA mini column (optional)
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Figures

Videos

Literature Cited

Literature Cited
   Boom, R., Sol, C.J., Heijtink, R., Wertheim‐van Dillen, P.M., and van der Noordaa, J. 1991. Rapid purification of hepatitis B virus DNA from serum. J. Clin. Microbiol. 29:1804‐1811.
   Boom, R., Sol, C., Beld, M., Weel, J., Goudsmit, J., and Wertheim‐van Dillen, P. 1999. Improved silica‐guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha‐casein to silica particles. J. Clin. Microbiol. 37:615‐619.
   Delfour, C., Roger, P., Bret, C., Berthe, M.L., Rochaix, P., Kalfa, N., Raynaud, P., Bibeau, F., Maudelonde, T., and Boulle, N. 2006. RCL2, a new fixative, preserves morphology and nucleic acid integrity in paraffin‐embedded breast carcinoma and microdissected breast tumor cells. J. Mol. Diagn. 8:157‐169.
   Greer, C.E., Lund, J.K., and Manos, M.M. 1991. PCR amplification from paraffin‐embedded tissues: Recommendations on fixatives for long‐term storage and prospective studies. PCR Methods Appl. 1:46‐50.
   Greer, C.E., Wheeler, C.M., and Manos, M.M. 1994. Sample preparation and PCR amplification from paraffin‐embedded tissues. PCR Methods Appl. 3:S113‐S122.
   Harada, S. and Gocke, C.D. 2010. Pathology case review: Specimen identity testing using DNA analysis in clinical and surgical pathology setting. Pathology Case Reviews. 15:116‐120.
   Laakso, S., Kirveskari, J., Tissari, P., and Mäki, M. 2011. Evaluation of high‐throughput PCR and microarray‐based assay in conjunction with automated DNA extraction instruments for diagnosis of sepsis. PLoS. One. 6:e26655.
   Mirmomeni, M.H., Sajjadi Majd, S., Sisakhtnezhad, S., and Doranegrad, F. 2010. Comparison of the three methods for DNA extraction from paraffin‐embedded tissues. J. Biol. Sci. 10:261‐266.
   Moelans, C.B., Oostenrijk, D., Moons, M.J., and van Diest, P.J. 2011. Formaldehyde substitute fixatives: Effects on nucleic acid preservation. J. Clin. Pathol. 64:960‐967.
   Morikawa, T., Shima, K., Kuchiba, A., Yamauchi, M., Tanaka, N., Imamura, Y., Liao, X., Qian, Z.R., Brahmandam, M., Longtine, J.A., Lindeman, N.I., Fuchs, C.S., and Ogino, S. 2012. No evidence for interference of H&E staining in DNA testing: Usefulness of DNA extraction from H&E‐stained archival tissue sections. Am. J. Clin. Pathol. 138:122‐129.
   Murphy, K.M., Berg, K.D., Geiger, T., Hafez, M., Flickinger, K.A., Cooper, L., Pearson, P., and Eshleman, J.R. 2005. Capillary electrophoresis artifact due to eosin: Implications for the interpretation of molecular diagnostic assays. J. Mol. Diagn. 7:143‐148.
   Regan, J.F., Furtado, M.R., Brevnov, M.G., and Jordan, J.A. 2012. A sample extraction method for faster, more sensitive PCR‐based detection of pathogens in blood culture. J. Mol. Diagn. 14:120‐129.
   Srinivasan, M., Sedmak, D., and Jewell, S. 2002. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am. J. Pathol. 161:1961‐1971.
   Turashvili, G., Yang, W., McKinney, S., Kalloger, S., Gale, N., Ng, Y., Chow, K., Bell, L., Lorette, J., Carrier, M., Luk, M., Aparicio, S., Huntsman, D., and Yip, S. 2012. Nucleic acid quantity and quality from paraffin blocks: Defining optimal fixation, processing and DNA/RNA extraction techniques. Exp. Mol. Pathol. 92:33‐43.
   Verheyen, J., Kaiser, R., Bozic, M., Timmen‐Wego, M., Maier, B.K., and Kessler, H.H. 2012. Extraction of viral nucleic acids: Comparison of five automated nucleic acid extraction platforms. J. Clin. Virol. 54:255‐259.
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