new polymerases for old dna: molecular breeding of polymerases for damage bypass and ancient dna...

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. 2001 2:353

The directed evolution cycle

Ghadessy et al (2001) PNAS, 98, 4552

Oil phase

Water droplet

Crameri et al (1998) Nature, 391, 288Zhao 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

Taq Tth Tfl

3A10

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

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

PCR amplification of ancient cave bear DNA

Exp Samplea Dilution Taq PCR Blend PCR Improvement 1 GS3-7 1/500 24 / 36 28 / 36 +16% 2 GS3-7 1/2000 2 / 24 5 / 24 +150% 3 GS3-7 1/1000 21 / 48 24 / 48 +14% 4 366 1/5 2 / 4 4 / 4 +100% 5 366 1/10 12 / 24 16 / 24 +33% 6 GS3-7 1/1000 12 / 48 14 / 48 +16% 7 GS3-7 1/1000 10 / 24 7 / 24 -30% 8 GS3-7 1/1000 12 / 48 13 / 48 +8% 9 GS3-7 1/1000 12 / 48 13 / 48 +8% Total 107 / 304 124 / 304 15%

366 derives from the Herdengel cave (Austria) (ca. 60 kyrs old); GS3-7 derives from the Gamsulzen cave (Austria) (ca. 47 kyrs old)

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)

106 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’AbbadieFarid J. GhadessyDavid Loakes

Max Planck Institute for Molecular Anthropology

Michael HofreiterSvante Pääbo

NIH Section on DNA Replication, Repair and Mutagenesis

Alexandra VaismanRoger Woodgate