Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Note
  • Published:

Use of quantitative polymerase chain reaction analysis to compare quantity and stability of isolated murine DNA

Lab Animal volume 39, pages 283–289 (2010)Cite this article

Abstract

Deoxyribonucleic acid (DNA) can be extracted from different tissue sources for genotyping of mice. Methods of collecting tissue vary with respect to their perceived invasiveness, and in some cases, tissue is collected multiple times in order to verify a genotype. The authors' goal was to refine and optimize tissue collection methods with quantitative measures for subsequent genotyping. To do this, the authors used real-time quantitative polymerase chain reaction (qPCR) analysis to quantify DNA extracted from fecal pellets, hair, buccal swab samples, ear punch samples and tail biopsy samples and then compared the quantities of DNA obtained by using either previously published protocols or commercial kits to extract DNA. They found that 2-mm tail biopsy samples yielded significantly more DNA than did the other sources and that commercial extraction kits generally yielded more DNA than did published protocols. The authors also assessed the stability of DNA extracted from the various tissue sources by repeating the qPCR analysis after the samples had been frozen and stored for 44 months. Although the quantities of DNA in the stored samples had decreased, all the samples could still be used for PCR-based genotyping. The authors' work supports the collection of a single minimal biopsy sample of 2 mm of tail or ear tissue, above other sources, for highest yield of DNA for genotyping. With appropriate storage, DNA remains usable for PCR-based genotyping for years without a need for repeated animal sampling.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Standard curves of DNA concentration relative to cycle threshold (C(t)) at site A and site B.
Figure 2: DNA quantity (μg) determined using quantitative qPCR at (a) site A (soon after DNA extraction) and (b) site B (44 months after DNA extraction).
Figure 3: Routine genotyping of qPCR products.

Similar content being viewed by others

References

  1. Pinkert, C.A. Transgenic animal technology: alternatives in genotyping and phenotyping. Comp. Med. 53, 126–139 (2003).

    CAS  PubMed  Google Scholar 

  2. Attal, J., Cajero-Juarez, M. & Houdebine, L.M. A simple method of DNA extraction from whole tissues and blood using glass powder for detection of transgenic animals by PCR. Transgenic Res. 4, 149–150 (1995).

    Article  CAS  Google Scholar 

  3. Burkhart, C.A., Norris, M.D. & Haber, M. A simple method for the isolation of genomic DNA from mouse tail free of real-time PCR inhibitors. J. Bioch. Biophys. Methods 52, 145–149 (2002).

    Article  CAS  Google Scholar 

  4. Drews, R., Drohan, W.N. & Lubon, H. Transgene detection in mouse tail digests. Biotechniques 17, 866–867 (1994).

    CAS  PubMed  Google Scholar 

  5. Gaw, A., Mancini, F.P. & Ishibashi, S. Rapid genotyping of low density lipoprotein receptor knockout mice using a polymerase chain reaction technique. Lab. Anim. 29, 447–449 (1995).

    Article  CAS  Google Scholar 

  6. Henneberger, C., Grantyn, R. & Rothe, T. Rapid genotyping of newborn gene mutant mice. J. Neurosci. Methods 100, 123–126 (2000).

    Article  CAS  Google Scholar 

  7. Lin, C.S., Magnuson, T. & Samols, D. A rapid procedure to identify newborn transgenic mice. DNA 8, 297–299 (1989).

    Article  CAS  Google Scholar 

  8. Petkov, P.M. et al. Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse. Genomics 83, 902–911 (2004).

    Article  CAS  Google Scholar 

  9. Petkov, P.M. et al. An efficient SNP system for mouse genome scanning and elucidating strain relationships. Genome Res. 14, 1806–1811 (2004).

    Article  CAS  Google Scholar 

  10. Sorensen, D.B. et al. The impact of tail tip amputation and ink tattoo on C57BL/6JBomTac mice. Lab. Anim. 41, 19–29 (2007).

    Article  Google Scholar 

  11. Zimmermann, K., Schwarz, H.P. & Turecek, P.L. Deoxyribonucleic acid preparation in polymerase chain reaction genotyping of transgenic mice. Comp. Med. 50, 314–316 (2000).

    CAS  PubMed  Google Scholar 

  12. Campbell, D.B. & Hess, E.J. Rapid genotyping of mutant mice using dried blood spots for polymerase chain reaction (PCR) analysis. Brain Res. Brain Res. Protoc. 1, 117–123 (1997).

    Article  CAS  Google Scholar 

  13. Chen, S. & Evans, G.A. A simple screening method for trangenic mice using the polymerase chain reaction. Biotechniques 8, 32–33 (1990).

    CAS  PubMed  Google Scholar 

  14. Gregory, C.A., Myal, Y. & Shiu, R.P. Rapid genotyping of transgenic mice using dried blood spots from Guthrie cards for PCR analysis. Biotechniques 18, 758–760 (1995).

    CAS  PubMed  Google Scholar 

  15. Hofstetter, J.R. et al. Genomic DNA from mice: a comparison of recovery methods and tissue sources. Biochem. Mol. Med. 62, 197–202 (1997).

    Article  CAS  Google Scholar 

  16. Irwin, M.H., Moffatt, R.J. & Pinkert, C.A. Identification of transgenic mice by PCR analysis of saliva. Nat. Biotech. 14, 1146–1148 (1996).

    Article  CAS  Google Scholar 

  17. Meldgaard, M., Bollen, P.J. & Finsen, B. Non-invasive method for sampling and extraction of mouse DNA for PCR. Lab. Anim. 38, 413–417 (2004).

    Article  CAS  Google Scholar 

  18. Mitrecic', D., Mavric', S., Branica, B.V. & Gajovic', S. Mice genotyping using buccal swab samples: an improved method. iochem. Genet. 46, 105–112 (2008).

    Article  CAS  Google Scholar 

  19. Lahm, H., Hoeflich, A., Rieger, N., Wanke, R. & Wolf, E. Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum. Transgenic Res. 7, 131–134 (1998).

    Article  CAS  Google Scholar 

  20. Broome, R.L. et al. Non-invasive transgenic mouse genotyping using stool analysis. FEBS Lett. 462, 159–160 (1999).

    Article  CAS  Google Scholar 

  21. Ohhara, M., Kurosu, Y. & Esumi, M. Direct PCR of whole blood and hair shafts by microwave treatment. Biotechniques 17, 726–728 (1994).

    CAS  PubMed  Google Scholar 

  22. Schmitteckert, E.M., Prokop, C.M. & Hedrich, H.J. DNA detection in hair of transgenic mice—a simple technique minimizing the distress on the animals. Lab. Anim. 33, 385–389 (1999).

    Article  CAS  Google Scholar 

  23. Busler, D.E. & Li, S.W. Rapid screening of transgenic type II and type XI collagen knock-out mice with three-primer PCR. Biotechniques 21, 1002–1004 (1996).

    Article  CAS  Google Scholar 

  24. Malumbres, M., Mangues, R., Ferrer, N., Lu, S. & Pellicer, A. Isolation of high molecular weight DNA for reliable genotyping of transgenic mice. Biotechniques 22, 1114–1149 (1997).

    Article  CAS  Google Scholar 

  25. Ren, S., Li, M., Cai, H., Hudgins, S. & Furth, P.A. A simplified method to prepare PCR template DNA for screening of transgenic and knockout mice. Contemp. Top. Lab. Anim. Sci. 40, 27–30 (2001).

    CAS  PubMed  Google Scholar 

  26. Cinelli, P., Rettich, A., Seifert, B., Bürki, K. & Arras, M. Comparative analysis and physiological impact of different tissue biopsy methodologies used for the genotyping of laboratory mice. Lab. Anim. 41, 174–184 (2007).

    Article  CAS  Google Scholar 

  27. Wilhelm, J. & Pingoud, A. Real-time polymerase chain reaction. Chembiochem 4, 1120–1128 (2003).

    Article  CAS  Google Scholar 

  28. Institute for Laboratory Animal Research. Guide for the Care and Use of Laboratory Animals (National Academies Press, Washington, DC, 1996).

  29. Schunck, B., Kraft, W. & Truyen, U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces. J. Virol. Methods 55, 427–433 (1995).

    Article  CAS  Google Scholar 

  30. Safadi, F.F. et al. Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein. J. Clin. Invest. 103, 239–251 (1999).

    Article  CAS  Google Scholar 

  31. Freeman, B. et al. DNA from buccal swabs recruited by mail: evaluation of storage effects on long-term stability and suitability for multiplex polymerase chain reaction genotyping. Behav. Genet. 33, 67–72 (2003).

    Article  CAS  Google Scholar 

  32. Hankenson, F.C., Garzel, L.M., Fischer, D.D., Nolan, B. & Hankenson, K.D. Evaluation of tail biopsy collection in laboratory mice (Mus musculus): vertebral ossification, DNA quantity, and acute behavioral responses. J. Am. Assoc. Lab. Anim. Sci. 47, 10–18 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Wilson, W.O., Riskin, D.K. & Mott, K.M. Ultrasonic sound as an indicator of acute pain in laboratory mice. J. Am. Assoc. Lab. Anim. Sci. 47, 8–10 (2008).

    Google Scholar 

Download references

Acknowledgements

We thank Jessica Knowlton (formerly of the UM Orthopedic Research Laboratories) and Shawn Terkhorn and Alice Huang (formerly of the University of Pennsylvania) for technical assistance. We thank the American College of Laboratory Animal Medicine Foundation and the Universities of Michigan and Pennsylvania for financial support. K.D.H. was supported by National Institutes of Health grants R01 AR49862 and K01 RR RR00161.

Author information

Author notes

  1. Laura M. Garzel and F. Claire Hankenson: These authors contributed equally to this work.

Authors and Affiliations

  1. University Laboratory Animal Resources, The Ohio State University, Columbus, OH

    Laura M. Garzel

  2. University Laboratory Animal Resources, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

    F. Claire Hankenson

  3. Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

    F. Claire Hankenson

  4. Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

    Jason Combs &  Kurt D. Hankenson

Authors
  1. Laura M. Garzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F. Claire Hankenson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jason Combs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kurt D. Hankenson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to F. Claire Hankenson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Garzel, L., Hankenson, F., Combs, J. et al. Use of quantitative polymerase chain reaction analysis to compare quantity and stability of isolated murine DNA. Lab Anim 39, 283–289 (2010). https://doi.org/10.1038/laban0910-283

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/laban0910-283

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing