Forensic Sci Med Pathol (2007) 3:221–225
DOI 10.1007/s12024-007-9009-5
DNA REVIEW
DNA reviews: Ancient DNA
E. A. M. Graham
Accepted: 18 July 2007 / Published online: 11 October 2007
Ó Humana Press Inc. 2007
Abstract The rapid development, success, and occa-
sional failures of forensic DNA profiling are highly
publicised, and as a consequence are well known to the
scientific and public communities alike. Over the same
period of time that forensic DNA typing has accelerated
onto the scene, another related discipline has been born and
has made equally, or perhaps, even more groundbreaking
achievements in the same arena; that is the science of
(bio)molecular archaeology. This review describes the
extreme complications of ancient DNA analysis and
highlights some of the major achievements that have been
accomplished in this field. The nature of DNA degradation
and possible solutions to this problem are discussed.
Keywords Forensic science
Molecular archaeology

Bone
DNA degradation
Mitochondrial DNA

MiniSTR
Introduction
Over the last two decades, the rise and rise of forensic
DNA profiling has taken the scientific, legal and media
communities by storm. From high-profile criminal cases
such as the trial of Simpson during 1995 that introduced
and indeed discredited the use of DNA in criminal inves-
tigation, to the more-recent quest to determine the
biological father of the late Anna Nicole Smith’s daughter,
Dannielynn, DNA profiling has been the focus of attention
E. A. M. Graham (&)
Forensic Pathology Unit, University of Leicester, Robert
Kilpatrick Building, Leicester Royal Infirmary, Leicester LE2
7LX, UK
e-mail: eamg1@le.ac.uk
for millions of people, and dollars. During this time, a
separate discipline has evolved in parallel, using many of
the same techniques employed in forensic DNA investi-
gation, which has also fueled the imagination of scientific
and public minds alike, ancient DNA analysis. The early
1990s saw the release of the blockbuster movie Jurassic
Park and the scientific publications that allowed us to
believe that sequencing of DNA millions of years old was
indeed possible [1, 2]. These early claims have since been
rejected due to the inability of independent laboratories to
reproduce the findings [3]. There have since however, been
numerous examples whereby ancient sequences have been
recovered, analysed and independently verified. The most
notable example of which being the sequencing of mito-
chondrial DNA from Neanderthal remains [4, 5].
Molecular archaeology
Molecular (a.k.a. biomolecular) archaeology can include
the analysis of any ancient biological material, defined as
being older than 75 years. As the most informative bio-
molecule, DNA has been the major focus of attention for
many practitioners of this discipline. The analysis of
ancient DNA (aDNA) was first reported during the mid-
1980s when mitochondrial DNA (mtDNA) sequences of
the extinct quagga (a zebra-like species) were sequenced
from tissue sampled from a 140-year-old museum piece
[6]. This was quickly followed by the report of nuclear
DNA recovery from 2,400-year-old tissue collected from
an Egyptian mummy [7]. At the same time Professor Sir
Alec Jeffreys was making the discovery that would forever
change forensic science, personal individualisation by
DNA fingerprinting [8, 9]. Although each of these works
was monumental in its own right, molecular biology was
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yet to enter the polymerase chain reaction (PCR) era. The
introduction of this technique revolutionised molecular
biology, with ancient and forensic DNA analysis perhaps
the most to gain from its invention [10]. Almost immedi-
ately, PCR, combined with advances in DNA extraction
procedures made primarily by Erika Hagleberg allowed the
analysis of nuclear DNA from ancient [11–13] and forensic
bone specimens [14].
DNA degradation
In order to understand the complexities involved in the
study of degraded DNA it is in many respects important to
have a comprehensive understanding of the chemical
structure and nature of the molecule. The primary structure
of DNA was first elucidated, using X-ray diffraction, by
Watson and Crick in 1953 [15]. They proposed the double-
stranded helical structure that is today a very familiar
image. The primary structure of DNA can be described as a
long-chained heteropolymer, with each monomer consist-
ing of three subunits: 20 deoxyribose, pentose sugar and
phosphate. One of four organic bases (adenine, guanine,
cytosine and thiamine) is then incorporated in this complex
to provide the sequence information. Bonding occurs
between the sugar and phosphate to form a nucleoside. This
bonding gives the DNA directionality (written as 50 ? 30 )
as a phosphate group is always present at the 50 terminus
with a hydroxyl group being present at the 30 terminus. The
informational units or organic bases are attached to the 10
carbon atom of the pentose ring via a b-N-glycosidic bond.
Organic bases are then joined via a hydrogen bond, to form
the familiar double-helix structure of DNA, which is
strengthened by base stacking and p–p interactions. The
helical DNA molecule is then wound around proteins
called histones, with 100–200 bp of DNA around each unit.
In an effort to protect the DNA from chemically induced
damage the protein-bound DNA is then supercoiled to form
chromatin. It is also worth mentioning at this point that the
structure of DNA in vivo is unstable. This is due to the
enthalpy of the chemical reaction, in an aqueous environ-
ment, which favours the hydrolysis of polynucleotides, to
allow DNA to take up a less-rigid, but thermodynamically
favourable, random conformation. A reoccurring concern
of many published works and review articles is that of the
degradation processes of DNA. The first and still most
significant work was produced in 1993 by Tomas Lindahl
in a paper entitled ‘Instability and decay of the primary
structure of DNA’ [16]. As well as DNA’s inherent insta-
bility there are many external factors that can contribute to
its degradation, the most influential are discussed below.
Post mortem DNA degradation begins immediately after
the death of the organism via the action of endogenous
Forensic Sci Med Pathol (2007) 3:221–225
nucleases, cellular enzymes that catalyse the hydrolysis of
phosphodiester bonds in the DNA backbone. The obser-
vation that recovered ancient and forensic DNA molecules
are usually between 100 bp and 200 bp in length has led to
the suggestion that this enzymatic cleavage occurs at vul-
nerable linker regions, situated between nucleosomes of the
DNA tertiary structure. It logically follows that, for the
survival of any DNA, the action of these biological
enzymes must be slowed or halted before complete deg-
radation is achieved. This reduction of enzymatic activity
is entirely dependent on the surrounding environmental
conditions. Burger et al. [17] attempted to address the
effect of varying environmental conditions on the preser-
vation of DNA. By taking bone samples of similar age but
greatly differing site milieu a direct comparison of DNA
survival and preservation conditions is achieved. The
environmental conditions of temperature, humidity, pH and
the geochemical properties of the soil were recorded. In
summary their findings indicate site aridity, low tempera-
ture, neutral or alkaline pH and the absence of destructive
and contaminating micro-organisms as the best DNA
preservation conditions [17].
The action of endogenous nucleases is not the only
factor affecting DNA preservation. The effect of hydrolysis
on the inherently weak b-N-glycosidic bond between the
organic base and the pentose sugar ring has a major
destructive effect on DNA. Hydrolysis has two main
effects on the DNA molecule, as discussed by Lindahl [16].
Firstly, acid-catalysed hydrolysis causes depurination and
to a lesser extent depyrimidination of DNA, causing the
loss of primary sequence information. Secondly, the
organic bases themselves are susceptible to hydrolytic
deamination causing instability in the DNA molecule as a
whole. Damage induced by oxidation is another important
consideration in DNA preservation. Oxidation has been
observed to cause the formation of saturated pyrimidine
rings, the loss of the double bond between carbon atoms 5
and 6, resulting in a noncoding base derivative. The
resulting modified bases are collectively known as hydan-
toins, molecules that are not recognised by Taq polymerase
and thus inhibit PCR [18]. It has been proposed that the
quantity of oxidatively modified bases present in a sample
of aDNA can provide a useful indicator of the likelihood of
amplification [19]. Base modifications were directly
quantified by gas chromatography/mass spectroscopy (GC/
MS) and an inverse correlation was observed between the
amount of oxidised pyrimidines (C and T) and the ability to
amplify aDNA.
Some other factors that must also be taken into account
include the presence of bacteria and fungi, as mentioned
above. These micro-organisms contribute to the degrada-
tion of DNA not only by digestion but also increase the risk
of contamination of the sample by extraneous DNA. The
Forensic Sci Med Pathol (2007) 3:221–225
presence of micro-organisms in samples is environmentally
influenced, with arid, cold and oxygen-deprived conditions
reducing the risk of infestation and corresponding to the
optimum DNA survival conditions noted above. The effect
of ultraviolet (UV) damage is another important consider-
ation, as UV radiation is known to form crosslinks between
thiamine residues, which are another inhibitor of PCR.
Inhibition of the PCR reaction is not confined to DNA
modifications; a naturally occurring acid was identified in
1994. It has been acknowledged that even trace amounts of
fulvic acid in a PCR reaction can severely reduce the
efficiency of Taq polymerase [20]. It has also been noted
that the presence of humic and fulvic acids in the envi-
ronment surrounding buried skeletal remains shows a
positive correlation with the general humidity of the area
[17]. Humidity and therefore warm temperature and
moisture, itself can accelerate DNA degradation by
allowing organic substances from sediments to penetrate
hard tissues such as bone and teeth.
Additional challenges
Contamination with contemporary DNA is the major issue
that has to be addressed when working with ancient DNA.
The efficiency of the PCR system is itself a major problem
in degraded DNA work as a PCR system has the potential
to initiate amplification from as little as a single molecule
of DNA. Although contamination is a risk when partaking
in any PCR-based investigation, when working with human
DNA the problem is even more acute. In the case of aDNA,
contamination arises from several main sources including,
handling of the remains by excavators, museum staff, as
well as airborne contaminants from the laboratory and
contaminants present in laboratory reagents or on con-
sumable items. To minimise the risk of amplifying
contaminating modern DNA from ancient specimens a
number of criteria have been developed [21–25] which
were summarised in the 2004 review of aDNA analyses by
Svante Pääbo et al. [26] and include the use of extraction
and amplification controls, repeated amplification of the
same or preferably several extracts for each sample,
quantitation of the number of amplifiable template mole-
cules present in DNA extracts, observation of an inverse
correlation between fragment length and amplification
efficiency and reproducibility in a second laboratory.
Even if all of the recommended criteria are adhered to
and contamination is not thought to have entered the PCR
reaction a multitude of problems can be encountered during
the PCR itself. The thermostable Taq polymerase used is
responsible for incorrect base insertion at a rate of one per
1,000 bases (C–T transitions). This is not usually a problem
as the erroneous base readings are extremely unlikely to
223
occur at the same position more than once so repeat
experiments and multiple sequencing can compensate for
this error. The phenomenon of jumping PCR induced by
damaged template DNA was first reported in 1990 [27].
Jumping occurs when Taq polymerase reaches the end of a
template molecule and inserts an adenosine, terminating
the extension. This molecule can then jump onto another
template and polymerisation continues, resulting in the
production of a chimeric sequence. This recombinant
molecule is then amplified in subsequent rounds of PCR
and thus will be present in large amounts by the end of the
reaction. In the majority of aDNA work the obtained
sequence is expected to be unique, which could lead the
investigator to believe that this chimeric DNA molecule is
the true sequence. Again this problem can be recognised by
multiple sequencing. It has been proposed that the recog-
nition of the jumping phenomenon can be used as a
measure of the authenticity of the amplified DNA [23].
This is based on the knowledge that jumping will only
occur if the template is severely damaged, as is expected in
the case of ancient DNA.
Future directions
Despite all of the difficulties encountered, aDNA analysis
has today become an almost routine undertaking, with
reports of successful amplification of woolly mammoth
[28], Chinese [29] and Siberian mummies [30], Amerin-
dian skeletons [31], ancient wheat seeds [32], ground sloth
coprolites [24, 33], and even dodo [34] DNA, to name but a
few. Despite their apparent separation, ancient and forensic
communities have occasionally combined to tackle cases
that reside on the border of the ancient—forensic temporal
boundary, perhaps the most famous example being the
identification of the Romanov family remains by both
mtDNA and short tandem repeat (STR) analysis [35]. But
despite some successful meetings, and calls for further
discussion [36] the two communities have remained largely
independent. One particular development in forensic DNA
profiling, which was highlighted in the first DNA Review
of this series [37], may once again bring the disciplines
together. MiniSTR kits under development by John But-
ler’s group at the National Institute of Standards and
Technology (NIST) [38] and the International Commission
on Missing Persons (ICMP) [39] as well as the recently
launched AmpFlSTR MiniFiler kit from Applied Biosys-
tems are now allowing forensic scientists to obtain STR
profile information from degraded template material which
previously would not have been possible. The rationale
behind the development of miniSTR kits is intentionally
directed towards the successful amplification of any
remaining short DNA fragments that can be recovered
224
from decomposed or skeletonised modern material; the
presence of PCR inhibitors in such samples has also been
taken into account. Modified PCR chemistries included in
such developments also facilitate amplification of DNA
even in the presence of such inhibitors. This technology
could also prove to be extremely useful in the analysis of
ancient human samples and this approach could be exten-
ded to the myriad of additional species of interest to the
molecular archaeologist.
Educational message
1. Ancient DNA analysis includes the investigation of
any biological material older than 75 years.
2. DNA degradation commences immediately after the
death of an organism and will continue at different
rates depending upon its environment.
3. Advancements in molecular biological technique have
allowed for the analysis of mtDNA from samples over
10,000 years old.
4. Recent developments in forensic DNA profiling may
allow for further information to be gained from ancient
samples.
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