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Related Experiment Videos

DNA damage promotes jumping between templates during enzymatic amplification.

S Pääbo1, D M Irwin, A C Wilson

  • 1Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.

The Journal of Biological Chemistry
|March 15, 1990
PubMed
Summary

DNA damage can cause polymerase chain reaction (PCR) primers to jump between templates, creating chimeric molecules. This "jumping PCR" phenomenon can lead to inaccurate sequencing of damaged DNA, like ancient DNA.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Polymerase chain reaction (PCR) is a fundamental technique for DNA amplification.
  • DNA damage, including breaks, apurinic sites, and UV damage, can affect DNA integrity.
  • Accurate DNA sequencing is crucial for genetic research, diagnostics, and ancient DNA studies.

Purpose of the Study:

  • To investigate the impact of DNA template lesions on PCR amplification.
  • To characterize the mechanism of primer extension termination and template switching during PCR.
  • To evaluate the consequences of these events on the accuracy of DNA sequencing, particularly for damaged templates.

Main Methods:

  • Designed specific primer-template pairs to detect recombination events during PCR.

Related Experiment Videos

  • Compared DNA sequences obtained through direct sequencing of PCR products versus sequencing after bacterial cloning.
  • Utilized the thermostable Thermus aquaticus DNA polymerase.

    • Main Results:

    • DNA lesions (breaks, apurinic sites, UV damage) induce primer 'jumping' to alternative templates during PCR.
    • Thermus aquaticus DNA polymerase can insert an adenosine residue upon encountering template ends, leading to premature termination.
    • This prematurely terminated product can switch templates, resulting in chimeric amplification products, especially from damaged DNA like ancient DNA.

    Conclusions:

    • PCR amplification of damaged DNA templates frequently produces chimeric molecules due to template switching.
    • Direct sequencing of PCR products can mask these chimeras, while cloning reveals illegitimate residues, potentially leading to misinterpretation.
    • The 'jumping PCR' phenomenon can be leveraged to quantify and map DNA lesions in nucleic acid samples.