Ancient DNA pp 111-119 | Cite as

PCR Amplification, Cloning, and Sequencing of Ancient DNA

  • Tara L. FultonEmail author
  • Mathias Stiller
Part of the Methods in Molecular Biology book series (MIMB, volume 840)


PCR amplification of DNA is routine in modern molecular biology. However, the application of PCR to ancient DNA (aDNA) experiments often requires significant modification to standard protocols. The degraded nature of most aDNA fragments requires targeting shorter fragments, performing replicate amplifications, incorporating multiple negative controls, combating PCR inhibition, using specific DNA polymerases to deal with damaged bases, working in a separate aDNA facility, and modifying the PCR recipe to deal with damaged and low copy-number target DNA. In this chapter, we describe how and why these procedures are implemented, discuss aDNA-specific troubleshooting methodology, and suggest modifications to commercial cloning and sequencing procedures to reduce the expense of PCR product cloning.

Key words

Polymerase chain reaction PCR optimization BSA, inhibition Ancient DNA DNA polymerase 


  1. 1.
    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle-cell anemia. Science 230:1350–1354PubMedCrossRefGoogle Scholar
  2. 2.
    Poinar HN, Schwarz C, Qi J, Shapiro B, MacPhee RDE, Buigues B, Tikhonov A, Huson DH, Tomsho LP, Auch A, Rampp M, Miller W, Schuster SC (2006) Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science 311:392–394PubMedCrossRefGoogle Scholar
  3. 3.
    Rohland N, Hofreiter M (2007) Comparison and optimization of ancient DNA extraction. Biotechniques 42:343–352PubMedCrossRefGoogle Scholar
  4. 4.
    Heyn P, Stenzel U, Briggs AW, Kircher M, Hofreiter M, Meyer M (2010) Road blocks on paleogenomes-polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA. Nucleic Acids Res 38:e161PubMedCrossRefGoogle Scholar
  5. 5.
    Willerslev E, Cooper A (2005) Ancient DNA. Proc Biol Sci 272:3–16PubMedCrossRefGoogle Scholar
  6. 6.
    Poinar HN, Hofreiter M, Spaulding WG, Martin PS, Stankiewicz BA, Bland H, Evershed RP, Possnert G, Paabo S (1998) Molecular coproscopy: dung and diet of the extinct ground sloth Nothrotheriops shastensis. Science 281:402–406PubMedCrossRefGoogle Scholar
  7. 7.
    Bartlett JMS, Stirling D (2003) PCR protocols, 2nd edn. Humana, Totowa, NJCrossRefGoogle Scholar
  8. 8.
    Sambrook J, Russell DW (2006) The condensed protocols from molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of BiologyThe Pennsylvania State UniversityUniversity ParkUSA

Personalised recommendations