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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bustin SA (2004) A-Z of quantitative PCR. International University Line, La Jolla, CA
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193
Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34:597–601
Rohland N, Hofreiter M (2007) Comparison and optimization of ancient DNA extraction. Biotechniques 42:343–352
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 277:1991–2000
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 104:739–744
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 35:1–10
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):e5
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 46:195–200
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 29:753–762
Bustin SA (2010) Why the need for qPCR publication guidelines?—The case for MIQE. Methods 50:217–226
Acknowledgments
MB was supported by the Australian Research Council as a Future Fellow (FT0991741). We thank Jayne Houston and James Haile for helpful discussions and Beth Shapiro for valuable editorial inputs.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Bunce, M., Oskam, C.L., Allentoft, M.E. (2012). Quantitative Real-Time PCR in aDNA Research. In: Shapiro, B., Hofreiter, M. (eds) Ancient DNA. Methods in Molecular Biology, vol 840. Humana Press. https://doi.org/10.1007/978-1-61779-516-9_16
Download citation
DOI: https://doi.org/10.1007/978-1-61779-516-9_16
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-515-2
Online ISBN: 978-1-61779-516-9
eBook Packages: Springer Protocols