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By Peter Malinski

Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA + Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae. By Peter Malinski. Woolly Mammoths.

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By Peter Malinski

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  1. Removal of deaminatedcytosines and detection of in vivo methylation in ancient DNA + Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae By Peter Malinski

  2. WoollyMammoths Mammoths are an extinct species that lived during the Pleistocene as recently as 10,000 years ago. Some mammoths have been recovered in remarkable condition in recent times which has led to scientific inquiry about the genetics and phylogeny of this creature. Even though ancient DNA has a plethora of inherent problems in replication, this scientific inquiry has advanced to the point of reproducing this creature’s entire mitochondrial genome.

  3. Ancient DNA • Ancient DNA isdefined as DNA extractad fromancientorganisms. (belonging to times long past especially of the historical period before the fall of the Western Roman Empire) • In studying the genomes of extinct species, two principal limitations are typically the small quantities of endogenous ancient DNA and it degraded condition • Under favorable conditions, DNA can survive in tissue remains for several millennia and in some cases over 100,00 years

  4. Deaminatedcytosines • DNA sequencesdeterminedfromancientorganismshavehigher error ratesprimarilyduetouracil bases createdbycytosinedeamination. • The reaction of a water molecule with the amino-group on position 4 of the pyrimidine ring of cytosine, which results in the conversion of cytosine to uracil.

  5. Can we reverse it? • Treatment of ancient DNA withuracil-DNA-glycosylase and endonuclease VIII removesuracilresidues and repairsmost of theresultingabasicsites, leavingundamagedparts of DNA fragments in tact.

  6. High-throughputDirectSequencing • High-throughputDirectSequencing can sequence DNA molecules of alllengths. In thisapproach, theends of ancient DNA fragments are madeamenabletoligationbytreatmentwith T4 DNA polymerase and T4 polynucleotidekinase. Subsequently DNA adaptors are ligatedtothefragmentends and then are usedtoamplify individual moleculeswhichinitiatesequencingreactionson a highlyparallelizedplatform.

  7. Methods • Take several different samples (synthetic oligonucleotides, mammoth DNA, neandertal DNA) and run them through the sequencing. • Standard blunt end repair • Damage repair, no CIP treatment • Damage repair, CIP-treatment • Ligation • Adaptor fill-in • Quantification and sequencing • Repair scheme: In library preparation protocols for high-throughput sequencing of ancient DNA, PNK and T4 DNA polymerase are used to generate ends amenable to DNA ligation. A large proportion of nucleotide misincorporations generated from ancient DNA libraries are caused by uracils present in short 5’overhangs. These are filled in during end repair reactions.

  8. Results: It worked. • The treatment was successful in all three cases

  9. PCR amplification of mammoth mitochondrial genome • Multiplex polymerase chain reaction allowed the entire mitochondrial genome to be amplified using just two initial amplifications accompanied by primer pairs that defined overlapping DNA sequence fragments.

  10. What happened? • 46 primer pairs were used to amplify the entire mtDNA. These primer pairs were combined into two sets, each containing every second primer pair. • The two primary amplifications are diluted and used as templates in secondary PCR reactions, in which each product was amplified individually.

  11. Phylogeny • Comparisons based on complete mtDNA sequences were made. • Absolute number of phylogenetically informative positions on which the three species test is based • Relative length of the internal branch in the two groups of species for clock like phylogenetic trees. • *molecular clock = uniform rate of nucleotide substitutions in all lineages.

  12. Conclusion • Ancient DNA of mammoths is now better understood than ever before. • Understanding evolutionary relationships (divergences between the mammoth and Asian elephant took place only 440,000 years ago) • Cloning possibilities. • Ethical considerations

  13. Sources • 1. Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae. Krause J, Dear P, Pollack J, Slatkin M, Spriggs H, Barnes I, Lister A, Ebersberger I, Paabo S, Hofreiter M. NautreVol 439 9 Feb. 2006 doi: 10.1038/nature04432 • 2. Removal of deaminatedcytosines and detection of in vivo methylation in ancient DNA. Briggs A, Stenzel U, Meyer M, Drause J, Kircher M, and Paabo S. Nucleic Acids Research, 2010, Vol. 38, No 6. 22 December 2009. • 3. Orlando L, Darlu P, Toussaint M, Bonjean D, Otte M, and Hanni C. (2006) Revisiting Neandertal diversity with a 100,000 year old mtDNA sequence. CurrIol., 16, • 4. Paabo S, et al. Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645-679 (2004). • 5. Tajima F. Simple methods for testing the molecular evolutionary clock hypothesis. Genetics 135, 599-607 (1993) • 6. Felsenstein, J. Confidence-limits on phylogenies with a molecular clock. Syst. Zool. 34, 152-161 (1985)

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