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
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.
Woolly Mammoths
Ancient DNA is defined as DNA extractad from ancient organisms. (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
Ancient DNA
Deaminated cytosinesDNA sequences
determined from ancient organisms have higher error rates primarily due to uracil bases created by cytosine deamination.
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.
Can we reverse it?Treatment of ancient
DNA with uracil-DNA-glycosylase and endonuclease VIII removes uracil residues and repairs most of the resulting abasic sites, leaving undamaged parts of DNA fragments in tact.
High-throughput Direct Sequencing
High-throughput Direct Sequencing can sequence DNA molecules of all lengths. In this approach, the ends of ancient DNA fragments are made amenable to ligation by treatment with T4 DNA polymerase and T4 polynucleotide kinase. Subsequently DNA adaptors are ligated to the fragment ends and then are used to amplify individual molecules which initiate sequencing reactions on a highly parallelized platform.
MethodsTake several different samples
(synthetic oligonucleotides, mammoth DNA, neandertal DNA) and run them through the sequencing.
Standard blunt end repairDamage repair, no CIP
treatmentDamage repair, CIP-treatmentLigationAdaptor 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.
Results: It worked.
The treatment was successful in all three cases
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.
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.
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.
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
Conclusion
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. Nautre Vol 439 9 Feb. 2006 doi: 10.1038/nature04432
2. Removal of deaminated cytosines 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. Curr Iol., 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,
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