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