DNA damage promotes jumping between templates during enzymatic amplification.
Pairs of templates and primers were designed so that only recombination events would lead to amplification via the polymerase chain reaction. This approach reveals that lesions such as breaks, apurinic sites, and UV damage in a DNA template can cause the extending primer to jump to another template during the polymerase chain reaction. By comparing sequences of amplification products that were determined directly or via bacterial cloning, it was shown that when the thermostable Thermus aquaticus DNA polymerase encounters the end of a template molecule, it sometimes inserts an adenosine residue; the prematurely terminated product then jumps to another template and polymerization continues, creating an in vitro recombination product. Consequently, amplification products from damaged templates such as archaeological DNA are made up of a high proportion of chimeric molecules. The illegitimate adenosine and thymidine residues in these molecules are detected when cloned molecules are sequenced, but are generally averaged out when the amplification product is sequenced directly. However, if site-specific lesions exist in template DNA or if the amplification is initiated from very few copies, direct sequencing also may yield incorrect sequences. The phenomenon of the "jumping polymerase chain reaction" can be exploited to assess the frequency and location of lesions in nucleic acids.
Pääbo
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Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Irwin
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Wilson
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D000241:Adenosine; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D009106:Mummies; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology
D004272:DNA, Mitochondrial; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000241:Adenosine