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Brief communication Minimally invasive bone sampling method for DNA analysis.

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AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 139:596–599 (2009)
Brief Communication: Minimally Invasive Bone Sampling
Method for DNA Analysis
Victoria E. Gibbon,1,2* Clem B. Penny,1 Goran Štrkalj,3 and Paul Ruff1
1
Department of Internal Medicine, University of the Witwatersrand, Johannesburg, Parktown,
Johannesburg 2193, South Africa
2
School of Geography, Archaeology and Environmental Studies, University of the Witwatersrand, Johannesburg,
Parktown, Johannesburg 2193, South Africa
3
Department of Health and Chiropractic, Macquarie University, Sydney, NSW 2109, Australia
KEY WORDS
ancient DNA; bone sampling; skeletal elements; temperature; drill; bone
ABSTRACT
Obtaining a bone sample for DNA analysis has traditionally been a destructive practice,
which has resulted in reluctance on behalf of curators
for skeletal collections to allow invasive testing. A
novel minimally invasive bone sampling method for
DNA analysis is presented here. This method uses a
conventional hand drill wherein the bone sample is
extracted from the intercondylar fossa of the femur; it
does not interfere with any known anthropometric
landmarks and only leaves a small hole on the surface
of the bone. The temperature of the drill is documented
and it was established due to the minor increase in
temperature, that this should not affect the molecular
integrity of the sample. This method is easily replicated and is suitable for both human and other animal
skeletal material and can be applied to rare specimens
with little risk. Am J Phys Anthropol 139:596–599,
2009. V 2009 Wiley-Liss, Inc.
In various anthropological, archaeological, and forensic
studies a detailed examination of skeletal remains is
conducted to reconstruct the demographic variables of
an individual or a population. The analysis of DNA
extracted from bone tissue has proved invaluable in this
process. However, extracting bone tissue from the skeleton has traditionally been destructive to the holistic
value of the skeleton (Kaestle and Horsburgh, 2002). A
section of or a whole bone is often removed and
destroyed (O’Rourke et al., 2000) leaving a skeleton with
missing elements or sections. In turn, this has led to reluctance by many researchers and curators of skeletal
material to allow such invasive procedures, specifically
on rare specimens.
A minimally invasive method of bone sampling is presented here that can successfully be applied to both fragmented skeletons and those of juveniles with intact
femora––materials that are difficult to assess using
traditional morphometric-based methods. In this study,
specific criteria were developed to determine the optimal
area on the skeleton to harvest a bone sample suitable
for DNA analysis in a minimally invasive way. The bone
sampling method should not interfere with any known
sites used in morphological or metrical analysis and the
skeletal element must not be destroyed. As bone preservation has been associated with DNA integrity (Haynes
and Searle, 2002), sources of and contaminant risks from
the environment infiltrating the skeletal element need to
be considered. Therefore, only whole skeletal elements
without any damage or lesions on the external surface,
or at the distal and proximal ends should be used.
Lastly, a skeletal element with a significant amount of
cancellous bone is recommended here, as it is protected
from environmental contamination (i.e. soil and roots) by
compact bone (Machugh et al., 2000; Wurmb-Schwark et
al., 2003) and is easier to pulverize.
THE AREA FOR THE BONE SAMPLING
C 2009
V
WILEY-LISS, INC.
C
It would appear that long bones (particularly the ends)
best satisfy the above outlined criteria for bone sampling. One of the bones often used is the femur, although
the exact place of the sampling is not always specified.
We propose that the best sampling site is the intercondylar fossa (Woodward et al., 2006; Gibbon, 2008), as this
area has no definitive features of identification and it is
not used in morphological or metrical analyses. In addition, there is a good store of cancellous bone at the distal
end of the femur.
THE SAMPLING TECHNIQUE
The sample must be mechanically broken down to a
powder form with minimal damage to the bone. Extra
manipulation increases the surface area for contaminant
DNA molecules to bind to, a factor that must be considered (O’Rourke et al., 2000). In considering the destruction to the skeletal material and to gain access for molecGrant sponsors: Palaeontological Scientific Trust (PAST), Medical
Research Endowment Fund (University of the Witwatersrand),
J. J. J. Smiezek Bursary.
*Correspondence to: Victoria Gibbon, Department of Internal
Medicine, University of the Witwatersrand, Medical School, 7 York
Rd, Parktown, Johannesburg 2193, South Africa.
E-mail: gibbonv@gmail.com
Received 29 September 2008; accepted 23 January 2009
DOI 10.1002/ajpa.21048
Published online 6 April 2009 in Wiley InterScience
(www.interscience.wiley.com).
MINIMALLY INVASIVE BONE SAMPLING METHOD
597
Methods
Fig. 1. Demonstration of the hole made in the intercondylar
fossa of the femur during the bone sampling procedure.
ular studies on valuable collections such as the Raymond
Dart Collection of Human Skeletons (Dart Collection), it
is important that the method be minimally destructive
and that it allows for the collection of future data.
A simple effective method of producing bone powder
was accomplished here through the use of a sterile titanium bit attached to a power drill. After drilling, the
bone powder is poured directly into a sterile container,
which decreases the opportunity for contamination. It
only forms a small hole on the surface (see Fig. 1) and
does not interfere with any known anthropometric landmarks. Although the use of this region ensures minimal
destruction to the bone, it also provides enough bony tissue for DNA analyses. This technique is only marginally
invasive, and does not physically compromise the
femur’s use in future studies. As the use of a power-drill
may increase the temperature in the bone that could
lead to DNA degradation, two experiments were carried
out to document the temperature emitted during this
procedure.
MATERIALS AND METHODS
Materials
The bone sampling method was optimized using material from the so-called X-collection housed in the School
of Anatomical Sciences, University of Witwatersrand,
Johannesburg. This collection consists of incomplete or
damaged cadaver-derived skeletal specimens that were
de-accessioned from the well-known Dart Collection.
Once devised, this method was applied to archaeological
skeletons sourced from the Dart Collection. Ethical clearance was obtained to use these specimens from the
School of Anatomical Sciences Collections Committee.
Strict contamination precautions were taken as prescribed for aDNA research (Hagelberg et al., 1989; Haas
et al., 2000; Machugh et al., 2000; O’Rourke et al., 2000;
Brown, 2001; Mays et al., 2002). Protective clothing was
worn, consisting of doctor’s scrubs, latex gloves, hairnet,
face mask, and booties (Hagelberg et al., 1991; Kolman
and Tuross, 2000; Cobb, 2002). Drill bits and 50 ml centrifuge tubes were autoclaved (Stericlav-28), in addition
a new bit and sterile preweighed tube were used for
each sample.
Within a dedicated area of the laboratory, the bone
sampling was performed under a sterilized laminar flow
hood to protect the working surfaces. The sampling area
was sterilized with 0.6% sodium hypochlorite followed by
70% ethanol (Kolman and Tuross, 2000; Wurmb-Schwark
et al., 2003, 2004a; Hebsgaard et al., 2005).
Under the flow form hood, paper toweling was placed
over the cleaned working surfaces. The entire surface of
the femur was first wiped down with 0.6% sodium hypochlorite followed by 70% ethanol (Kemp and Smith,
2005). The surface in the intercondylar fossa was
scraped with a scalpel to remove any contaminant DNA
from the surface, then cleaned with 70% ethanol (Hagelberg and Clegg, 1991; Zoledziewska et al., 2002; Holland
et al., 2003). The femur was held by the neck in the left
hand, in a vertical position, and a power drill (Bosch
PSB 650RE) with a sterile 4.5 mm titanium masonry
drill bit was used to create a hole between the lateral
and medial condyles in the intercondylar fossa. Once
through the cortical surface, a rotating action was used
to pulverize cancellous bone from the inner table. The
drill bit was withdrawn and the femur inverted over the
open mouth of a 50 ml tube. The bone powder was gently
tapped into the tube to collect approximately 1 g of bone
powder. The extracted bone powder was stored for a
short period of time at 48C. In between each sampling
gloves and surfaces were washed and sterilized with
0.6% sodium hypochlorite, followed by 70% ethanol.
Temperature Experiments
The temperature of the bone sampling procedure was
recorded through two experiments. The temperature in
both cases was measured using two copper-constantan
thermocouple probes connected to a laboratory digital
thermometer (Physitemp, Model BAT-12; Sensortek, Clifton, NJ) and was tested against a mercury thermometer.
On average a mercury thermometer took up to 15 s to
take a measurement, whereas the superior thermocouple
probe took only 3–5 s. Published data by the Sensortek
Inc. company is supportive of the accuracy of these
measurements. The wires were held in plastic forceps, as
not to transfer any heat from the researcher to the
probe. The temperature changes were assessed using
five femora.
In experiment one, prior to drilling the ambient temperature of both the drill bit and bone were recorded.
The temperature was also recorded on both the tip of the
drill bit and the edge of the cortical bone in the hole,
upon both the initial breakthrough of the cortical bone
and also during the pulverization.
In experiment two, a temperature probe was placed
inside a predrilled hole a few millimeters away from the
intercondylar fossa of the femur, and the ambient temperature was recorded. Then while drilling an adjacent
American Journal of Physical Anthropology
598
V.E. GIBBON ET AL.
TABLE 1. Recorded temperature data in this table in degrees
Celsius for experiment 1
Drill bit specimen no.
Before drilling
Initial breakthrough
Pulverize
Bone hole specimen no.
Before Drilling
Initial Breakthrough
Pulverize
1
2
3
4
5
18.7
20
19.4
18.7
19.2
19.3
18.8
19
19
18.8
19.4
19.3
18.9
19.4
19.8
18.4
18.8
19.1
18.4
18.4
18.8
18.6
18.8
18.8
18.5
18.8
18.9
18.4
18.9
19.2
hole in the intercondylar fossa (extraction site), the temperature was simultaneously recorded from within the
predrilled hole as described earlier.
RESULTS
The novel method described earlier was applied to 30
archaeological skeletons sourced from the Dart Collection. Access to this material was only granted after it
was shown that the method was minimally invasive on
skeletal material sourced from the X-collection. This
sample of 30 specimens produced an average yield of
0.77 g of fine to medium grained bone powder, which is
an ample amount for DNA sampling. Because of bone
mass there was individual variation in the amount of
bone yielded, which is most likely related to ageing.
Two experiments were setup to test the temperature
emitted from the drill for this newly developed method.
For the first experiment the change in temperature was
measured for initial breakthrough and during the pulverization procedure for both the drill bit and the bone.
The results are recorded in Table 1. The average
increase in temperature for initial breakthrough of the
cortical bone on the drill bit was 0.628C and for the hole
in the bone was 0.368C. For the pulverizing action, the
average increase in temperature of the drill bit was
0.588C and for the hole in the bone it was 0.428C. During
this experiment it was noted that the drill bit had the
greatest increase in temperature during the initial
breakthrough. On the other hand, the bone experienced
the greatest temperature change during the pulverization procedure.
In the second experiment, the temperature was
recorded from within the bone during the entire drilling
process. The temperature probe was placed inside an
existing hole and the temperature was recorded while
drilling and the measured temperatures are recorded in
Table 2. The change in average temperature recorded
both while and after drilling was 0.848C. One notable
feature of the change in temperature seen in both
experiments was the correlation of temperature increase
with the quality of the starting material. For bones
sourced from younger specimens with thicker cortical
bone and a high density of cancellous bone, the sampling
process takes slightly longer, thus increasing the temperature. The opposite result was found for specimens with
low bone density and thin cortical bone, there was virtually no change in temperature. The maximum change
from ambient temperature was 0.848C; therefore, it can
be stated with confidence that the temperature for this
drilling process does not reach excessively hot temperatures that would destroy the DNA. These temperature
American Journal of Physical Anthropology
TABLE 2. Recorded temperature data in this table in degrees
Celsius for experiment 2
Specimen no.
1
2
3
4
5
Before drilling
During drillinga
After drilling
19
19.8
19.7
18.9
19.5
19.5
18.9
19
19.1
18.7
19.3
19.3
18.6
20.7
20.7
a
The temperature was recorded for the drilling process with a
probe recording inside an adjacent hole.
variations are minimal in comparison to studies that
have extracted DNA from incinerated remains that have
endured temperatures upwards of 1508C (Urbani et al.,
1999; Wurmb-Schwark et al., 2004b).
This bone sampling method has been used in two studies using skeletal material for molecular sex determination (Gibbon, 2008). In each of these studies access to
the skeletal material for sampling was granted because
of the minimally invasive nature of the bone sampling
method.
DISCUSSION
The minimally invasive bone sampling methods available for molecular studies using skeletal tissue are not
always adequately described in the literature. However,
there are other minimally invasive techniques available,
some using drill-like instruments (Faerman and BarGal, 1998; Greenwood et al., 1999; Adcock et al., 2001).
Those that use drilling do so inconsistently and on different aspects of the skeleton. There are also minimally
invasive methods that use a buffered solution to dissolve
the bone (Asher and Hofreiter, 2006). The majority of
these methods are not used on human remains, and as
of yet there is no published method that takes into consideration damage to morphological and metrical areas
on the skeleton. As developed here, it is important to
have a minimally invasive sampling method from bone
that can be replicated in any laboratory.
CONCLUSION
The novel bone sampling method devised here is particularly important as it is minimally invasive to the
specimen and does not interfere with any anthropometric points of identification. It uses a drill and creates a
small hole in the intercondylar fossa of the femur,
retrieving enough bone powder from the internal surface
of the skeletal element for molecular studies. Because of
the minor change in temperature emitted during the
bone sampling process, this should not affect molecular
integrity of the obtained sample. This method of bone
sampling is easily replicated and applicable for molecular studies on both human and animal skeletal material.
It can be used to isolate mtDNA or any gene of interest
in the genome. Because of its minimally invasive nature,
this technique can be applied to rare specimens with little risk that permits the use of available collections for
genetic studies.
ACKNOWLEDGMENTS
We would like to thank Dr. Kathleen Kuman for her
support in the preparation of this manuscript. We would
also like to thank Miss Robyn Hetem for providing us
with the necessary temperature probes for this study.
MINIMALLY INVASIVE BONE SAMPLING METHOD
We would also like to thank Dr. James Lanoway for his
valuable input.
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