Analysis of Radiocarbon, Stable Isotopes and DNA in Teeth to Facilitate Identification of Unknown Decedents
Stable Isotopes and DNA in Teeth to Facilitate
Identification of Unknown Decedents. PLoS ONE 8(7): e69597. doi:10.1371/journal.pone.0069597
Analysis of Radiocarbon, Stable Isotopes and DNA in Teeth to Facilitate Identification of Unknown Decedents
Bruce A. Buchholz
David R. Senn
Kirsty L. Spalding
Lyle Konigsberg, University of Illinois at Champaign-Urbana, United States of America
The characterization of unidentified bodies or suspected human remains is a frequent and important task for forensic investigators. However, any identification method requires clues to the person's identity to allow for comparisons with missing persons. If such clues are lacking, information about the year of birth, sex and geographic origin of the victim, is particularly helpful to aid in the identification casework and limit the search for possible matches. We present here results of stable isotope analysis of 13C and 18O, and bomb-pulse 14C analyses that can help in the casework. The 14C analysis of enamel provided information of the year of birth with an average absolute error of 1.861.3 years. We also found that analysis of enamel and root from the same tooth can be used to determine if the 14C values match the rising or falling part of the bomb-curve. Enamel laydown times can be used to estimate the date of birth of individuals, but here we show that this detour is unnecessary when using a large set of crude 14C data of tooth enamel as a reference. The levels of 13C in tooth enamel were higher in North America than in teeth from Europe and Asia, and Mexican teeth showed even higher levels than those from USA. DNA analysis was performed on 28 teeth, and provided individual-specific profiles in most cases and sex determination in all cases. In conclusion, these analyses can dramatically limit the number of possible matches and hence facilitate person identification work.
Competing Interests: The authors have declared that no competing interests exist.
The identification of unknown bodies is crucial for ethical,
medico-legal and civil reasons. Police and forensic investigators
worldwide spend considerable time every year attempting to solve
such cases. Typically, unidentified bodies or human remains are
eventually identified by fingerprint comparisons, comparison of
ante- and postmortem dental or medical radiographs, or by DNA
analysis of tissue from the dead body and from possible relatives.
However, in cases where there are no clues as to the identity, a
characterization of the body can limit and focus the search for
possible matches and help to exclude impossible or unlikely
alternatives. The Doe Network Database (http://www.
doenetwork.org) is extensively used in North America and includes
both reported missing persons and unidentified bodies. This
database contains information on about 3,800 unidentified
deceased individuals, the majority from the United States. A
careful review of this database (Table 1) showed that the estimated
age for adult individuals displayed an average uncertainty range of
15612 years. Such a wide range is not very helpful in the efforts to
limit the search for possible matches. Furthermore, the sex was
unknown or uncertain in some of the cases, and the geographic
origin of the person rarely known, apart from information about
the place where the victim was found. Hence, a more detailed
characterization of the subjects in terms of age, sex and
geographical origin is expected to improve the success rate in
dead victim identification work.
The most common procedure to estimate the age of an
unidentified body is to perform an anthropological examination,
which regarding skeletonized bodies focuses on age-related
changes in teeth and bones. However, anthropological methods
are not very precise and typically an error of 610 years is added to
the estimated age . Additional methods have therefore been
developed. Today, the most precise method to determine the age
at death is aspartic acid racemization analysis, which is based on
the observation that remaining aspartic acid in the tooth trapped
during its formation will be converted at a very slow rate from the
L-form to the D-form, both of which can be detected and
quantified by gas chromatographic methods . These methods
applied on enamel, dentin or cementum can provide an estimate
of the age of a person with a fairly good precision; using dentin the
error may be 65 years or less [3,4,5]. All the methods used to
determine age are based on age-dependent alterations in human
No. of Cases
Male (%) Asian (%)
N/A (%) Avg. low1 Avg. high1 Error range2
.1 yr 3 (%)
1The average minimum and maximum estimated age of the dead body.
2The average possible age range for all cases, where such estimation is provided.
3This figure represents the number of cases that still are unidentified after more than a year.
tissues, and hence they will all give an estimate of the persons age
at death. In contrast, the recently developed method to analyze
bomb-pulse 14C in enamel indicates the year of birth regardless of
when, or at what age, the person died . This method should not
be confused with the Libby method  to date archeological
material, which is based on the radioactive decay of 14C in
biological material. Instead, the bomb-pulse 14C method takes
advantage of the substantial increase in global 14C levels caused by
above-ground nuclear test bomb detonations 19551963
[8.9.10,11,12]. Repeated measurements of 14C in the atmosphere
and in biological products of known age, has over time resulted in
reference values to which analytical results can be compared to
offer an estimate of the age; see http://calib.qub.ac.uk/
Similarly, an important factor for limiting the search for possible
matches is the sex of the individual. Unknown human bodies that
are fairly well preserved rarely pose any problem, but if the
forensic case involves a mutilated or skeletonized body,
anthropological examination of bones that show sex dimorphism is often
performed. DNA profiling of bones and teeth is possible, and can
include markers for sex. However, if a fire victim is charred,
extraction and amplification of DNA from soft tissues and bones
can be difficult. Teeth represent the most resistant tissue in the
body and DNA analysis of teeth may be an alternative. Such
analysis, including markers for sex, has successfully been
performed even on incinerated bodies .
To limit the search for possible matches, the geographic origin
of the deceased may also provide clues to the identity. In birds,
analysis of stable isotopes in feathers formed during the winter has
been used to determine wintering areas . In a recent study,
hair samples from subjects living on different continents displayed
geographically specific isotopic signatures . In a previous
study, we have shown that such geographic differences regarding
13C also can be detected in teeth . This isotope is incorporated
into the tissues of animals, including humans, in relation to the
content in food. Since the diet by tradition varies geographically,
and is based on different primary products derived from plants
with different 13C content, the amounts in tissues will vary
accordingly . The geographic differences can be explained as
follows. Some types of plants can discriminate between 12C and
13C, resulting in differences in the levels of 13C between different
types of plants. C4 plants (which include corn and sugar cane)
contain higher amounts of 13C than C3 plants (which include
potato, wheat and sugar beet) since these strive to maximize their
CO2 assimilation. In general, C4 plants tend to grow in hotter or
drier climates than C3 plants. This in turn means that animals,
including humans, having a diet based mainly of C4 plants and/or
on animals that eat C4 plants, will incorporate more 13C than
those mainly living off C3 plant based diets.
18O is another stable isotope that shows geographic variation.
The incorporation of 18O in animal tissues is correlated to the
levels in drinking water and these levels vary with latitude because
of differences in the evaporation and condensation propensity
between 16O and 18O . In the United States, reports indicate
that the highest tap water 18O concentrations are in the south and
southeast and the lowest in the northwest . These data are
based on tap water samples collected from 20022003. Due to
differences in precipitation and variable ground water supplies to
specific areas, these levels may differ somewhat from year to year.
Still, 18O levels are not expected to change dramatically over time
and hence levels in teeth, bones and hair should mirror the levels
in tap water fairly well.
In this study we investigated whether analysis of bomb-pulse
derived 14C levels in teeth from subjects in North America
(Table 2) shows similar precision as in previous studies on teeth
from other continents. In addition, we analyzed 14C in both
enamel and roots of the same teeth to find out if this measure
could help to discriminate values that relate to incorporation
during the rising or falling part of the bomb-curve (Table 3). We
also wanted to explore possible geographical variation in 13C
(Table 4) and 18O (Table 5) by collecting teeth from Mexico,
Canada and different parts of the United States. From the same
teeth, we also performed DNA analysis of the amelogenin gene to
determine the sex of the subjects. Finally, the average enamel 14C
incorporation time in different types of teeth from this study were
combined with the results from previous studies [5,6,16] in order
to provide a reference guide for determining the date of birth of
persons (Table 6).
Date of Birth (DOB) Estimation Using Tooth Enamel
For all 66 teeth, sufficient amounts of enamel were obtained to
allow for 14C determination by accelerator mass spectrometry
(AMS). Table 2 displays the overall results and Table S1 provides
more detailed information. Nine of the teeth had a calculated
enamel formation time before 1955 and all showed pre-bomb 14C
values. For teeth laid down after 1955, a high correlation was
found between 14C levels in enamel and the actual formation time
of the enamel, with an average absolute error of 1.861.3 years,
R2 = .9935 (n = 57). The error of the estimates obtained using
Nolla  and crude 14C, respectively are given in Table S2, and
is further described in detail in Supporting online information.
Table S3 in Supporting Information S1 shows a comparison
between the Nollas calibration and crude 14C measurement in age
estimation. Table S4 in Supporting Information S1 provides
standard deviation in age at tooth formation for each tooth type
and jaw. The confidence intervals for the enamel DOB based on
14C incorporation time are given in Table S5 in Supporting
Information S1. In 14 cases, two or more teeth were analyzed
from the same individual. On average the mean difference in
estimates of date of birth of the person between teeth from the
same individuals was 1.4 (median 1.2) years. In Case 30, 14 teeth
had been extracted. The 14C values of each of these teeth
predicted the true DOB very well and the difference in prediction
of DOB between these teeth was small (Table 2). In nine of the
tooth DOB2 DOB
Table 2. Cont.
Tooth formation in/collected
No. time (yrs)1 in
tooth DOB2 DOB
1Enamel formation time according to Nolla .
2Average age of carbonate in dental enamel based on 14C measurement.
3Unclear at what age this subject moved to WA.
twelve cases, where the enamel was formed after the onset of the
bomb pulse, enamel 14C values matched the expected order of
formation, i.e. teeth with later formation time showed more recent
14C levels. In Cases 8 and 19 both teeth from each individual
showed very similar 14C values since both tooth types had similar
enamel formation times (Table 2). In Case 30, high 14C values
were seen in all teeth, which was in line with the fact that their
actual formation dates were during the peak of the bomb curve.
14C Incorporation in Tooth Roots
We also analyzed 14C levels in the roots from 28 teeth collected
from 26 individuals (Table 3). The 14C levels were consistently
higher in the roots than in the enamel if the enamel was formed
during the rising part of the curve, and lower in the roots if the
enamel was formed during the falling part of the curve. One
exception was Case 36, where the enamel was formed shortly
before the peak of the bomb-spike and most likely the lower level
of 14C in the root is explained by lower levels incorporated during
the falling part of the curve. In all pre-bomb cases, i.e. when the
enamel was completely formed before the bomb pulse, post-bomb
values were found in their corresponding roots, indicating that a
turnover in some component of the root continues even into old
age, as evidenced by the elevated 14C concentration in a man born
in 1918 (Case 2). This finding confirms previous observations of a
continuous formation of secondary dentin and cementum
throughout life [21,22].
Reference Levels of 14C to Determine Date of Birth
The principle for DOB determination of persons using 14C has
been based on subtracting the enamel formation time for the
particular type of tooth based on radiographic mapping of tooth
development (Fig. 1). However, in Table S2, we have compiled the
enamel DOB as estimated by 14C levels in this and previous
studies [5,6,16] and compared the result for each type of tooth
with the actual DOB of the person. We show that the error in birth
dating can be reduced by subtracting the average enamel 14C
incorporation time instead.
13C in Teeth can Tell Geographic Origin
13C levels in the tooth enamel from 41 teeth (33 individuals)
were analyzed with isotopic ratio mass spectrometry (Table 4).
The teeth extracted from subjects raised in North America showed
higher enamel 13C values compared to levels previously seen in
teeth collected from other continents, including South America
. The levels were somewhat lower in teeth from California,
British Columbia and Connecticut, compared to teeth from Texas.
Enamel from teeth from Mexico showed very high levels, even
higher than levels in enamel from teeth from the United States and
Canada, without overlap. 13C levels in 31 tooth roots from 27
individuals were also analyzed; 19 of the roots were from the same
teeth in which 13C enamel levels also were measured (Table 4).
The 13C levels in the roots were generally lower than in the
enamel. Furthermore, the 13C levels in tooth roots were higher in
Case Tooth Actual enamel
No. No. DOB
may explain the modest differences between states. Furthermore,
levels of 18O are also dependent on diet composition, since a large
part of the oxygen incorporated in tissues derives from food that
may be produced at remote locations.
A small part of the root of 29 teeth from 25 individuals was used
for DNA analysis. A full profile was obtained for 22 of the teeth
and in all cases the sex could be determined using the amelogenin
gene as a marker , see Fig. 3. In four cases, two teeth were
analyzed and the results were identical for different teeth from the
same individual. Furthermore, in select cases, the results of the
amelogenin gene analysis was confirmed by analysis of Y
chromosome specific STR markers using PowerplexH Y System
(Promega Corp., Madison, WI).
We report that 14C analysis of enamel from teeth collected from
North American subjects predicts the date of birth of the
individual with an average absolute error of 1.861.3 years. This
precision is similar to that of previous studies on teeth obtained
from other countries around the world [6,23,24,25,26]. Given that
a large number of above ground detonations were performed in
the Nevada desert, a concern regarding the reliability of this
method could be raised about subjects born and raised during the
cold war in nearby regions, such as California, Texas and northern
Mexico. However, from Table 2 and Table S1, it can be
appreciated that the precision of birth dating such individuals was
not different than that of others in this study. The enamel
formation times differ between different types of teeth, but also
show a variation among the same type of tooth, with third molar
teeth showing the highest variation [20,27]. Despite this fact, we
found that analysis of two or more teeth, including third molars,
from the same individual provided similar estimates of date of
birth of the individual. We also report that the 14C levels of two
teeth from the same individual disclosed the order in which the
teeth were formed, provided that their enamel formation times
were sufficiently different. This can help to differentiate between
teeth formed during the rising and falling part of the bomb-curve.
However, using one single tooth, we also show that 14C analysis of
both enamel and root can accomplish the differentiation. This
confirms the results that Cook et al. (2006) reported by analysis of
14C in enamel and root collagen [25,26], but shows that extraction
of the collagen constitutes an unnecessary step. Recently,
KondoNakamura et al. (2010) described a different approach, using
separate analysis of the occlusal and cervical part of the enamel of
the same tooth . However, they only describe such analysis of
two teeth, and the difference in 14C levels in the occlusal and
cervical samples were very small. It is therefore more reliable to
use either two teeth with sufficiently different enamel formation
times or the enamel and root from the same tooth for 14C analysis
to determine if the tooth has been formed during the rising or
falling part of the bomb-curve. In addition, cutting off the root
from the crown is much easier then separating different parts of
By analyzing teeth formed before the bomb spike we could show
that there is some turnover of carbon in the root after formation
has been completed. Since we only measured the mineral fraction
in the roots, the incorporation of 14C at adult ages in these roots
shows that this component of the dentin and/or cementum
continue to remodel, albeit at a slow pace given the relatively low
14C levels found. From Table 3 it can further be appreciated that
the teeth formed during the rising part of the curve (except Case
teeth from Mexico than in roots from the United States and
Canada, but showed a higher variation, making it more difficult to
separate Mexican subjects from persons raised in United States
and Canada using roots only.
Tap Water Provides an Additional Signature
In Table 5, the 18O levels in all roots analyzed are displayed.
18O levels were in general lower in tooth roots from the
northwestern region of North America (Washington and British
Columbia) compared to those in roots collected from Texas
(p,0.01). One subject from Alabama and one subject from Texas
were outliers, but otherwise the levels of 18O paralleled the tap
water levels fairly well (Fig. 2). Mexican teeth showed slightly lower
values than teeth from Texas and were similar to the average for
all teeth from the United States and Canada. From Table 5, it can
be appreciated that there are regional differences in the tooth
levels of 18O, but at a lower magnitude compared to the difference
in tap water levels. Since we did not collect information about the
exact place where the subjects were raised, interstate differences
1Enamel formation time according to Nolla .
36) have higher levels of 14C in the root than in the enamel,
implying that if a continuous turnover were significant, the
incorporation of the more recent atmospheric levels would have
produced lower 14C levels in the roots. In Case 36, the actual
enamel formation time was calculated to be 1961.3, i.e. shortly
before the atmospheric 14C maximum. Given the long time period
during which the roots are developed (completed about 67 years
after the enamel ), most of the incorporation of 14C would
have occurred during the falling part of the curve, explaining a
lower 14C level in the root than in the enamel.
The principle of estimating a persons date of birth from 14C
analysis of teeth has hitherto used reference data on tooth
Raised in/collected in
generate geographical signatures. Similarly, we have recently
reported that such differences can also be seen in tooth enamel
. In the present study we also show that 13C levels in tooth
enamel obviously vary within a limited geographical region such as
the United States and Canada. It seems likely that analysis of
several stable isotopes in teeth and hair can help to determine both
the earlier origin and more recent residence of an unknown dead
body and thus facilitate identification work. Interestingly, the 13C
levels in teeth from subjects raised in Mexico are the highest we
have recorded; even higher than in subjects from Chile and
Uruguay. The most likely explanation for this is a high dietary
intake of foods based on either corn or sugar cane, or both, since
they represent C4 plants that have double fixation steps in the
photosynthesis system and fail to discriminate between 13C and
12C like C3 plants such as wheat, potato, rice and sugar beet
[17,28]. The 13C levels in tooth roots (Tables 4 and 5) were
somewhat lower than in enamel from the same teeth and showed a
higher variation within restricted regions, making it less reliable
than enamel levels to determine geographic origin of the person.
This might be explained by a more traditional diet with locally
typical basic nutritional food sources during childhood than the
diet during adolescence.
The 18O levels in tooth roots were lowest in the northwest of the
United States, which is in accordance with the map of levels in tap
water previously reported , but showed somewhat variable
levels in other parts of the United States (Fig. 2). 18O levels in body
tissues may also be dependent on food intake with a variable level
of this isotope, given that a substantial part of the diet is composed
of water. Such contribution to the 18O levels in tooth enamel may
explain the smaller variation in teeth as compared to the variation
in tap water, since the different components of diet may come
from places at a distance from the residence - but perhaps not
sufficiently remote to make a difference regarding 13C.
The DNA analyses indicated the correct sex in all samples
analyzed. The sex determination is important to separate possible
matches from missing persons lists, but also for determining the
year of birth of the person, since this calculation is based on tooth
formation estimates and the tooth formation times are somewhat
different between sexes for several types of teeth. DNA analyses
are very swift and can be performed at short notice by many
laboratories worldwide. The consistent results using two different
methods for sex determination in select cases support the notion
that these analyses are robust. In addition, since both the
IdentifilerTM kit and the PowerplexH Y System provide a specific
profile of the individual, such an analysis will often be needed
anyway if antemortem radiographs cannot be retrieved and it
seems practical and more economical to perform this analysis
sooner than later. The Doe network database includes a large
number of unidentified persons and different identification
methods may be used to compare the characteristics of the
deceased with antemortem material from the deceased or with
samples from relatives. The review of this database (Table 1)
revealed that the estimates of age of the subject show a great
variation and that the sex was sometimes unknown. The analysis
of bomb-pulse derived 14C provides an accurate estimate of the
year of birth of the person, but only the age at death when the year
of death is known. We have previously shown that addition of
established methods to determine the age at death, such as aspartic
acid racemization, to the year of birth as estimated by 14C
measurement of teeth can be used to estimate the year of death of
the person .
In conclusion, from one single tooth, the year of birth can be
estimated, a clue to geographical origin can be obtained, sex can
be determined and analysis of 14C in both enamel and root can
1Unclear at what age this subject moved to WA.
formation, in turn based on repeated radiographs of children .
However, we have now compiled sufficient 14C results to calculate
an average age of carbon incorporation for most types of teeth to
bypass this step, so therefore we provide a reference tabulation of
average 14C incorporation times in Table S2 to be used for future
calculations. These estimates show a generally shorter time
interval than the radiographic results, which may be due either
to our choice of Nollas stage 4.5 as an average of the development,
or explained by incorporation into enamel of some of the carbon
earlier than the average radiographic picture of tooth. The
radiocarbon that we measure with AMS is that incorporated in the
carbonate component of the hydroxyapatite, and how this relates
to the degree of radio-opacity is not known. Nonetheless, the use of
crude 14C levels in the enamel of teeth from subjects with a known
DOB simplifies the calculation and obviously provides a more
precise better precision of the DOB estimate of unknown
individuals (Table S2).
Studies on hair samples from subjects in different US states 
and in different countries  have shown that stable isotopes
Absolute average error
using enamel formation time1
Absolute average error
using 14C incorporation time2
1Enamel formation time according to Nolla .
2The time interval used for error calculation does not include lag of 14C in the food chain.
differentiate between tooth formation during rising and falling part
of the bomb-curve. We have also previously reported that a small
portion of a tooth can be used for aspartic acid racemization to
estimate the age at death of a person, which will allow for a
calculation of the year of death, an important factor when
investigating a skeletonized body.
In conclusion, a better characterization of unknown dead
victims using these analyses can improve the identification work,
whether it concerns a suspect homicide case or victims of a mass
Materials and Methods
Collection of Teeth
In total, dentists in Mexico, the United States and Canada
collected 66 teeth. The teeth were either extracted for orthodontic
purposes or removed due to periodontal problems. Patients
permission to use the teeth for research instead of discarding
them was obtained in all cases and the study was approved by the
Regional Ethics Committee at the Karolinska Intitute (No 2010/
314-31/3). The tooth number, the year and month of birth, the
sex, the area where the person was raised, and the date of
extraction were noted and each person and tooth were given a
code. All teeth were shipped to Karolinska Institute for
preparation. A control determination of the tooth number was performed
blindly with the assistance of three independent forensic
Teeth were divided by cutting away the crown of the tooth from
the root at the level of the cervical line. To isolate the enamel, the
excised crown was immersed in 10 N NaOH and placed in a
water-bath sonicator overnight (Branson 150) at a maximum
temperature of 70uC. Approximately every 24 hrs the NaOH was
replaced and the softened non-enamel structures were removed by
mechanical treatment using a dental drill and blunt dissection.
Next, the enamel was washed in DDH2O, re-submersed in 10 N
NaOH and returned to the sonicator. This procedure was
repeated for 35 days until all dentin and soft structures were
completely removed. The enamel was then rinsed several times in
DDH2O and dried at 65uC overnight. The enamel was weighed
and kept sealed in a glass tube until pre-preparation for accelerator
mass spectrometric (AMS) analysis. Roots were vertically divided
to produce a somewhat larger piece for isotope analysis and a
smaller piece for DNA analysis. The roots were then rinsed in
DDH2O and no efforts were made to clean the root canal or to
Pre-treatment for AMS and Stable Isotope Mass
Aliquots of the enamel samples were placed in culture tubes for
pre-treatment to remove the surface carbon that may have
contaminated the enamel between formation and analysis.
Aliquots of 80150 mg were used to get full sized samples for
14C analysis. Enamel samples were immersed in 1.0 N HCl at
room temperature for 1.5 hrs, rinsed 3 times with DDH2O and
placed on a heating block at 95uC to dry overnight. The acid
pretreatment was designed to etch the outer surface of the enamel that
was exposed to the harsh base environment earlier without
dissolving too much of the enamel. Each dried enamel sample was
broken into 510 pieces and placed in an individual single-use
reactor. Enamel splits in individual reaction chambers were
evacuated, heated and acidified with concentrated
orthophosphoric acid at 90uC to hydrolyze all mineralized carbon to CO2. Roots
were treated with 2% NaOCl for 24 h, then rinsed 10 times with
DI water, followed by treatment with 0.1 N HCl for 30 min, again
rinsed 10 times with DI water and finally dried on a heating block
overnight. 120 mg of the roots were then pulverized, and 12 mg
of the powder was used for stable isotope analysis of 13C and 18O.
Stable oxygen and carbon isotope ratios were determined by
reaction of the powdered aliquot with supersaturated
orthophosphoric acid at 90uC in an Isocarb common acid bath
autocarbonate device attached to a Fisons Optima isotope ratio mass
spectrometer. This method liberates carbonate so only the mineral
fraction of the roots was used for stable isotope analysis.
The remainder of the powdered roots was used for AMS
analysis of 14C and underwent similar processing as enamel after
being placed in individual reactors. The evolved CO2 from roots
and enamel was purified, trapped, and reduced to graphite in the
presence of iron catalyst in individual reactors [29,30]. The CO2
from large enamel samples was split prior to graphitization and
d13C measured by stable isotope ratio mass spectrometry.
Background values were controlled by consistently following
procedures, frequently baking sample tubes, periodically cleaning
rigs, and maintaining a clean lab .
Accelerator Mass Spectrometry Analysis
Graphite targets were measured using the 10-MV HVEE
FNclass tandem electrostatic AMS system at the Center for
Accelerator Mass Spectrometry at Lawrence Livermore National
Laboratory (LLNL). The operation is similar to that used when
performing high-precision measurements of 18,000 year old
turbidities used as secondary standards . The system employs
a LLNL designed high-output negative ion solid graphite
Cssputter source  which emits 250350 mg of 12C- from a
fullsized sample, corresponding to approx. 900 14C counts per second
from a contemporary sample. The FN AMS system routinely
achieves 15% total system efficiency for C analyzing 14C4+ in the
detector . Details on the design of the LLNL AMS system and
its operation can be found in the literature [32,33,34,35]. Enamel
samples are usually full sized and contemporary, so analysis times
are relatively rapid, generally less than 5 minutes. The enamel
samples are measured for 30,000 14C counts per cycle for 57
cycle repetitions and achieve standard deviations of 0.30.8%.
Corrections for background contamination introduced during
AMS sample preparation are made by establishing the
contributions from contemporary and fossil carbon, following the
procedures of Brown and Southon . All data are normalized
using six identically prepared NIST SRM 4990B (Oxalic Acid I)
primary standards. NIST SRM 4990C (Oxalic Acid II), IAEA-C6
, and TIRI  wood are used as secondary standards and
quality controls to monitor spectrometer performance. The ratio
of NIST SRM 4990C to NIST SRM 4990B (Oxalic Acid II/
Oxalic Acid I) measured between February 2005 and March 2012
on 31 different sample wheels containing enamel samples had an
average value of 1.29160.003 (1 SD), in agreement with the
certified value of 1.29360.001. 14C-free calcite serves as
background material for processing the enamel samples. The
enamel samples are organized in groups of 1014 unknowns
bracketed by primary standards with one primary standard in the
middle of the group. The secondary standards, primary standards
and group of unknowns are measured consecutively as a cycle.
Upon completion of a cycle the set of primary standards,
secondary standards and unknown samples are measured again
until desired precision is achieved. A typical group of 14 enamel
samples is measured completely in 23 h. The measurement error
is determined for each sample and generally ranges between
60.20.8% (1 SD). All 14C data are reported using the F14C
fraction modern nomenclature developed for post-bomb data .
F14C is a concentration unit (14C/C) denoting enrichment or
depletion of 14C relative to oxalic acid standard normalized for
isotope fractionation. Data are also reported as decay corrected
D14C following the nomenclature of Stuiver and Pollach .
D14C was calculated using the equation:
where l = 1/8267 yr21 and y = year of measurement after
From 14C to Year of Birth
The average age at which enamel formation is completed for each
specific tooth has been determined previously and is dependent on
the specific tooth and sex of the person [20,27]. In cases where the
sex is unknown, the average time for enamel completion for males
and females can be calculated. However in this study the sex was
known for all individuals and confirmed by DNA analysis. To
estimate an individuals date of birth the 14C concentration
measured in the tooth enamel was compared with the Calibomb
reference data (http://calib.qub.ac.uk/CALIBomb) or Levin and
Kromer  to find out the year of enamel formation of the
particular tooth. By subtracting the average formation time for each
type of tooth , the individuals date of birth can be calculated.
If it is not obvious whether an individual is born before or after
the peak of atmospheric 14C levels resulting from the bomb tests,
then two teeth with different enamel formation times can be
analyzed this will distinguish whether the 14C measurement
relates to the rising or falling part of the curve. In 14 cases, two or
more teeth from the same individual were subjected to AMS
analysis. In addition, roots from the same teeth were also analyzed.
The root of a tooth will have been formed later than the enamel
and thus analysis of one single tooth can also provide such
information [25,27]. This principle is illustrated in Fig. 1.
DNA was extracted from small fragments of the 30 roots of
teeth according to a previously described method  for bone
DNA extraction. In short: the roots were placed in 96% ethanol
for a few minutes and rinsed with 0.5% Na-hypochlorite and dried
overnight at 56uC in open tubes. The roots were then ground to a
fine powder in liquid N2 in a Freezer/Mill 6850-115 (SPEX
Certiprep, New Jersey). The grinding cycles were the same as for
bone treatment, but grinding was shortened to only half a minute.
DNA was extracted from the total amount of tooth powder using
one phenol-chlorophorm and one chlorophorm extraction ,
concentrated on Centricon 30 columns (Millipore, Billerica, MA),
purified using the Qiaquick Purification Kit (Qiagen, Hilden,
Germany) and eluted in 45 mL buffer.
The DNA concentration was measured with a NanoDropH
2000 (Thermo Fischer Scientific, Wilmington, USA). Duplicate
aliquots of approximately 1 ng were used for the PCR
amplifications according to the IdentifilerTM protocol (Applied
Biosystems, Foster City, CA) in a final volume of 10 mL. To verify the
results in weak samples these were also amplified with a two-phase
PCR protocol of 10 and 20 cycles as previously described .
The DNA profiles, including amelogenin, the marker for the
determination of gender , were analyzed by capillary
electrophoreses in an ABI3100 Genetic Analyzer (Applied
Biosystems) and evaluated using GeneMapper ID 3.2 (Applied
Biosystems). In select cases, the results of the amelogenin gene
analysis was confirmed by analysis of Y chromosome specific STR
markers using PowerplexH Y System (Promega Corp., Madison,
WI), carried out according to the manufacturers manual. The
DNA extraction and amplification was performed using the same
PCR procedure as outlined for the IdentifilerTM protocol.
All results are given as means 6 SD. Differences between two
groups were examined for statistical significance with two-tailed
Students t test or with ANOVA, when appropriate. Significance
was accepted at a p value of ,.05.
The study was approved by the Regional Ethics Committee at
the Karolinska Intitute (No 2010/314-31/3). The ethics
committee approved this procedure provided that the patient or the
caretaker gave verbal informed consent. Since dental clinics have a
heavy workload, we declared that written consent would be
difficult to obtain, and the ethics committee agreed. There was no
documentation in the dental records that the extracted teeth were
used for analysis instead of being discarded. Only the year and
month of birth, and the gender were noted on the bags in which
the teeth were placed.
Table S1 Background data and all d 14C and d 13C
results for enamel.
Table S2 Compilation of DOB estimation using average
14C incorporation time interval only and using both 14C
and enamel laydown data.
Supporting Information S1 Supporting file containing
Tables S3S5. Table S3. Mean difference between
14Cmeasured age and Nollas calibration. Positive values indicate
that the measured age at tooth formation is larger than Nollas
estimate. Most values are negative, indicating that the
incorporation of radiocarbon on average is taking place earlier than the
enamel radio-opacity according to Nollas stage 4.5 estimate.
Table S4. Standard deviation in age at tooth formation for each
tooth type and jaw. Table S5. Confidence intervals for the date of
birth given the enamel formation year. Note that this does not use
Nollas estimates, only the 14C calibrated values.
All dentists procuring teeth are deeply acknowledged as well as the forensic
odontologists assisting in the confirmation of tooth type, in particular Irena
Dawidson and Hakan Bengtsson. Work was performed in part under the
auspices of the U.S. Department of Energy by Lawrence Livermore
National Laboratory under Contract DE-AC52-07NA27344.
Conceived and designed the experiments: KA. Performed the experiments:
KA HS BB SB GH. Analyzed the data: KA HS BB SB GH DS HD. Wrote
the paper: KA DS KS HD. Prepared the teeth and isolated enamel and
dentin: KA. Performed DNA extraction and DNA profiling: GH.
Collected teeth from dental clinical: DS.
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