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.
 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
 DNA 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 aminogroup on position 4 of the
pyrimidine ring of
cytosine, which results in
the conversion of cytosine
to uracil.
 Treatment of ancient
DNA with uracil-DNAglycosylase 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 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.
 Take several different samples
 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.

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(synthetic oligonucleotides,
mammoth DNA, neandertal
DNA) and run them through
the sequencing.
Standard blunt end repair
Damage repair, no CIP
treatment
Damage repair, CIPtreatment
Ligation
Adaptor fill-in
Quantification and
sequencing
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.
 The treatment was successful
in all three cases
 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.
 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.
 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

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
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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.
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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,
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4. Paabo S, et al. Genetic analyses from ancient DNA. Annu. Rev. Genet. 38, 645-679
(2004).
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5. Tajima F. Simple methods for testing the molecular evolutionary clock hypothesis.
Genetics 135, 599-607 (1993)
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6. Felsenstein, J. Confidence-limits on phylogenies with a molecular clock. Syst. Zool. 34,
152-161 (1985)