Molecular breeding of polymerases for amplification of ancient DNA.
In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.
d'Abbadie
Marc
M
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Hofreiter
Michael
M
Vaisman
Alexandra
A
Loakes
David
D
Gasparutto
Didier
D
Cadet
Jean
J
Woodgate
Roger
R
Pääbo
Svante
S
Holliger
Philipp
P
D004247:DNA / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics*; D016172:DNA Fingerprinting / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D053803:Forensic Genetics / Q000379:methods; D005580:Fossils; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
D004247:DNA; D004259:DNA-Directed DNA Polymerase