Ancient DNA pp 121-132

Part of the Methods in Molecular Biology book series (MIMB, volume 840) | Cite as

Quantitative Real-Time PCR in aDNA Research

  • Michael Bunce
  • Charlotte L. Oskam
  • Morten E. Allentoft
Protocol

Abstract

Quantitative real-time PCR (qPCR) is a technique that is widely used in the field of ancient DNA (aDNA). Quantitative PCR can be used to optimize aDNA extraction methodologies, to detect PCR inhibition, and to quantify aDNA libraries for use in high-throughput sequencing. In this chapter, we outline factors that need to be considered when developing efficient SYBR Green qPCR assays. We describe how to setup qPCR standards of known copy number and provide some useful tips regarding interpretation of qPCR data generated from aDNA templates.

Key words

qPCR Ancient DNA SYBR Green PCR inhibition qPCR standard DNA extraction optimization Library quantitation 

References

  1. 1.
    Bustin SA (2004) A-Z of quantitative PCR. International University Line, La Jolla, CAGoogle Scholar
  2. 2.
    Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193PubMedCrossRefGoogle Scholar
  3. 3.
    Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34:597–601PubMedCrossRefGoogle Scholar
  4. 4.
    Rohland N, Hofreiter M (2007) Comparison and optimization of ancient DNA extraction. Biotechniques 42:343–352PubMedCrossRefGoogle Scholar
  5. 5.
    Oskam CL, Haile J, McLay E, Rigby P, Allentoft ME, Olsen ME, Bengtsson C, Miller GH, Schwenninger JL, Jacomb C, Walter R, Baynes A, Dortch J, Parker-Pearson M, Gilbert MT, Holdaway RN, Willerslev E, Bunce M (2010) Fossil avian eggshell preserves ancient DNA. Proc Biol Sci 277:1991–2000PubMedCrossRefGoogle Scholar
  6. 6.
    Pruvost M, Schwarz R, Correia VB, Champlot S, Braguier S, Morel N, Fernandez-Jalvo Y, Grange T, Geigl EM (2007) Freshly excavated fossil bones are best for amplification of ancient DNA. Proc Natl Acad Sci USA 104:739–744PubMedCrossRefGoogle Scholar
  7. 7.
    Gilbert MT, Binladen J, Miller W, Wiuf C, Willerslev E, Poinar H, Carlson JE, Leebens-Mack JH, Schuster SC (2007) Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis. Nucleic Acids Res 35:1–10PubMedCrossRefGoogle Scholar
  8. 8.
    Meyer M, Briggs AW, Maricic T, Hober B, Hoffner BH, Krause J, Weihmann A, Paabo S, Hofreiter M (2008) From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing. Nucleic Acids Res 36(1):e5PubMedCrossRefGoogle Scholar
  9. 9.
    Allentoft M, Schuster S, Holdaway R, Hale M, McLay E, Oskam C, Gilbert MT, Spencer P, Willerslev E, Bunce M (2009) Identification of microsatellites from an extinct moa species using high-throughput (454) sequence data. Biotechniques 46:195–200PubMedCrossRefGoogle Scholar
  10. 10.
    Allentoft ME, Bunce M, Scofield RP, Hale ML, Holdaway RN (2010) Highly skewed sex ratios and biased fossil deposition of moa: ancient DNA provides new insight on New Zealand’s extinct megafauna. Quat Sci Rev 29:753–762CrossRefGoogle Scholar
  11. 11.
    Bustin SA (2010) Why the need for qPCR publication guidelines?—The case for MIQE. Methods 50:217–226PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Michael Bunce
    • 1
  • Charlotte L. Oskam
    • 1
  • Morten E. Allentoft
    • 1
  1. 1.Ancient DNA Laboratory, School of Biological Sciences and BiotechnologyMurdoch UniversityPerthAustralia

Personalised recommendations