New polymerases for old DNA: molecular breeding of polymerases for damage bypass and ancient DNA amplification Philipp Holliger MRC Laboratory of Molecular Biology

Mt. Vesuvius (Naples, Italy)Villa dei Papyrii Herculaneum Charcoal?ancient scroll ancient text The “unreadable” scrolls of Herculaneum

Ancient DNA Neanderthal man (Homo neanderthaliensis) our closest extinct relative PCR (1993 Nobel prize in Chemistry)

Damage found in ancient DNA From Hofreiter et al Nat Rev Genet :353

The directed evolution cycle

Ghadessy et al (2001) PNAS, 98, 4552 Oil phase Water droplet

Crameri et al (1998) Nature, 391, 288 Zhao et al (1998) Nature Biotechnology, 16,258 Molecular breeding

Selection strategy d’Abbadie et al (2007) Nature Biotechnology, 25, 939

Non-cognate primer-template structures d’Abbadie et al (2007) Nature Biotechnology, 25, 939

Damage bypass d’Abbadie et al (2007) Nature Biotechnology, 25, 939

PCR amplification of DNA containing two abasic sites TaqTthTfl 3A10 d’Abbadie et al (2007) Nature Biotechnology, 25, 939

PCR amplification of ancient cave bear DNA

d’Abbadie et al (2007) Nature Biotechnology, 25, 939

Cave bear (Ursus spelaeus) Wolly mammoth (Mammuthus primigenius) Mt genome (Krause et al Nature (2006),439, 724; Poinar et al Science (2006) 311, 392) Noonan et al, Science (2005), 309, 597 Neanderthal man (Homo neanderthaliensis) 10 6 bp sequenced: Green et al Nature (2006) 444, 330; Noonan et al Science (2006) 314, 1113 Ice age genomics

Acknowledgements MRC Laboratory of Molecular Biology Marc d’Abbadie Farid J. Ghadessy David Loakes Max Planck Institute for Molecular Anthropology Michael Hofreiter Svante Pääbo NIH Section on DNA Replication, Repair and Mutagenesis Alexandra Vaisman Roger Woodgate