Comparing different post mortem human samples as DNA sources for downstream genotyping and identification

Prévia do material em texto

Accepted Manuscript
Title: Comparing different post mortem human samples as
DNA sources for downstream genotyping and identification
Author: Gayvelline C. Calacal Dame Loveliness T. Apaga
Jazelyn M. Salvador Joseph Andrew D. Jimenez Ludivino J.
Lagat Renato Pio F. Villacorta Maria Cecilia F. Lim Raquel
d.R. Fortun Francisco A. Datar Maria Corazon A. De Ungria
PII: S1872-4973(15)30051-X
DOI: http://dx.doi.org/doi:10.1016/j.fsigen.2015.07.017
Reference: FSIGEN 1395
To appear in: Forensic Science International: Genetics
Received date: 23-3-2015
Revised date: 10-7-2015
Accepted date: 21-7-2015
Please cite this article as: G.C. Calacal, D.L.T. Apaga, J.M. Salvador, J.A.D.
Jimenez, L.J. Lagat, R.P.F. Villacorta, M.C.F. Lim, R.R. Fortun, F.A. Datar, M.C.A.D.
Ungria, Comparing different post mortem human samples as DNA sources for
downstream genotyping and identification, Forensic Science International: Genetics
(2015), http://dx.doi.org/10.1016/j.fsigen.2015.07.017
This is a PDF file of an unedited manuscript that has been accepted for publication.
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http://dx.doi.org/doi:10.1016/j.fsigen.2015.07.017
http://dx.doi.org/10.1016/j.fsigen.2015.07.017
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Comparing different post mortem human samples as DNA sources for downstream 1 
genotyping and identification 2 
 3 
Gayvelline C. Calacal
1,2
, Dame Loveliness T. Apaga
2
, Jazelyn M. Salvador
1,2
, Joseph Andrew D. 4 
Jimenez
3
, Ludivino J. Lagat
3
, Renato Pio F. Villacorta
4
, Maria Cecilia F. Lim
5
, Raquel dR. 5 
Fortun
5
, Francisco A. Datar
6
 and Maria Corazon A. De Ungria
1,2
 6 
 7 
1
DNA Analysis Laboratory, Natural Sciences Research Institute, University of the Philippines, 8 
Diliman, Quezon City, Philippines; 9 
2
Program on Forensics and Ethnicity, Philippine Genome Center, National Science Complex, 10 
University of the Philippines, Diliman, Quezon City, Philippines; 11 
3
Forensic Center, Commission on Human Rights, Central Office, Philippines;
 12 
4
Department of Anatomy, College of Medicine, University of the Philippines, Manila, 13 
Philippines;
 
14 
5
Department of Pathology, College of Medicine, University of the Philippines, Manila, 15 
Philippines;
 16 
6
Department of Anthropology, College of Social Science and Philosophy, University of the 17 
Philippines, Diliman, Quezon City, Philippines 18 
 19 
 20 
Additional Information and Reprint Requests: 21 
Gayvelline C. Calacal, RMT, MSc 22 
DNA Analysis Laboratory, Natural Sciences Research Institute 23 
University of the Philippines Diliman 24 
Quezon City 1101, Philippines 25 
gcalacal@gmail.com 26 
 27 
 28 
 29 
 30 
 31 
Title Page (with Author Details)
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 DNA analysis of several samples from various postmortem and environmental conditions 
 Complete DNA profiles were generated from bone marrow, femur, metatarsal and patella 
 Amplifiable DNA can be generated from bone marrow transferred on FTA
®
 card 
 Amplifiable target DNA maybe obtained using 0.1 ng DNA template 
 0.5 ng DNA template increased allele recovery and improved peak balance 
*Highlights (for review)
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Abstract 1 
 2 
The capability of DNA laboratories to perform genotyping procedures from post mortem remains, 3 
including those that had undergone putrefaction, continues to be a challenge in the Philippines, a 4 
country characterized by very humid and warm conditions all year round. These environmental 5 
conditions accelerate the decomposition of human remains that were recovered after a disaster 6 
and those that were left abandoned after a crime. When considerable tissue decomposition of 7 
human remains has taken place, there is no other option but to extract DNA from bone and/or 8 
teeth samples. Routinely, femur shafts are obtained from recovered bodies for human 9 
identification because the calcium matrix protects the DNA contained in the osteocytes. In the 10 
Philippines, there is difficulty in collecting femur samples after natural disasters or even human-11 
made disasters, because these events are usually characterized by a large number of fatalities. 12 
Identification of casualties is further delayed by limitation in human and material resources. 13 
Hence, it is imperative to test other types of biological samples that are easier to collect, transport, 14 
process and store. 15 
 16 
We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, 17 
femur, metatarsal, patella, rib and vertebral samples from five (5) recently deceased untreated 18 
male cadavers and seven (7) male human remains that were embalmed, buried for ~1 month and 19 
then exhumed. The bodies had undergone different environmental conditions and were in various 20 
stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by 21 
an organic procedure was used. The utility of bone marrow and vitreous fluid including bone 22 
marrow and vitreous fluid that was transferred on FTA
®
 cards and subjected to autosomal STR 23 
and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or 24 
absence of PCR inhibitors in DNA extracts was assessed using Plexor
®
HY. All samples were 25 
amplified using PowerPlex
®
21 and PowerPlexY
®
23 systems and analyzed using the AB3500 26 
Genetic Analyzer and the GeneMapper
®
 ID-X v.1.2 software. 27 
 28 
PCR inhibitors were consistently detected in bone marrow, muscle tissue, rib and vertebra 29 
samples. Amplifiable DNA were obtained in a majority of the samples analyzed. DNA recovery 30 
from 0.1 g biological material was adequate for successful genotyping of most of the non-bone 31 
and bone samples. Complete DNA profiles were generated from bone marrow, femur, metatarsal 32 
and patella with 0.1 ng DNA template. Using 0.5 ng DNA template resulted in increased allele 33 
recovery and improved intra- and inter- locus peak balance. 34 
*Manuscript
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Keywords: DNA, human identification, mass disasters, STR typing, putrefied remains 35 
 36 
1. Introduction 37 
DNA typing of samples such as bone and teeth that are obtained from putrefied remains is used 38 
for human identification after a disaster, during criminal investigations and in resolving parentage 39 
disputes involving deceased persons [1-7]. In the Philippines that is characterized by a tropical 40 
and humid climate and the presence of about 20 typhoons per year, proper management and 41 
processing of human remains for identification is important. For example, Typhoon Haiyan that 42 
greatly affected Eastern Visayas in 2014 resulted in at least 6,300 casualties [8]. Many bodies 43 
were already decomposed when washed ashore thereby making identification via visual, 44 
pathological and DNA examinations extremely difficult. Due to the magnitude of the tragedy, 45 
deficient logistic and financial resources and the compromised states of the remains, majority of 46 
the bodies were buried without proper identification. The complex political situation in the 47 
Philippines associated with an increased number of human-made casualties, e.g. bombing, and 48 
extra-judicial killings. From July 2010 to June 2014, there were 204 victims of extrajudicial 49 
killing [9]. Many bodies and body partsthat were found in abandoned locations or makeshift 50 
graves could not be identified using ordinary means. Hence in these situations, DNA profiling of 51 
putrefied remains or skeletonized samples may be the only means to identify their human sources. 52 
With the developments in DNA-based parentage testing amongst the living, many persons have 53 
likewise resorted to the collection of biological samples from deceased persons with the purpose 54 
of resolving parentage or kinship issues [10]. Many post-mortem samples that are submitted for 55 
DNA typing have been exposed to varied environmental conditions. These conditions may be 56 
associated with the embalming process when formalin (10% formaldehyde) is introduced into the 57 
bodies of the deceased, the exposure to lime commonly used to inhibit the decomposition process 58 
in mass graves; and contact with soil microorganisms after underground internment. Genotyping 59 
of post-mortem samples were reported unsuccessful due to low DNA yield, the presence of 60 
inhibitors in DNA preparations and DNA fragmentation/degradation brought about by exposure 61 
to harsh environmental conditions and microbial nucleases (1,3,4,11). 62 
 63 
There is a higher success rate of DNA recovery from femur shafts and teeth [3, 11]. Unlike 64 
spongy bones, the physical and chemical structure of a compact bone with the calcium matrix 65 
provides greater protection for DNA against post-mortem damage [11]. Several studies 66 
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recommended the collection of bone samples from the densest cortical bone especially when 67 
handling old skeletal remains for human identification [2, 6, 11]. In the Philippines, there is 68 
difficulty in collecting femur samples after natural disasters such as typhoons or even human-69 
made disasters such as bombings, because these events are usually characterized by a large 70 
number of fatalities. Identification of casualties is further delayed by limitation in human and 71 
material resources. Hence, it is imperative to test other types of biological samples that are easier 72 
to collect, transport, process and store. 73 
 74 
In the present study, four non-bone (vitreous fluid, bone marrow from clavicle, bone marrow from 75 
femur and muscle tissues) and six bone (femur, rib clavicle, vertebra, patella and metatarsal) type 76 
samples from recently deceased male persons and male remains with a short one-month post-77 
mortem interval were genotyped using autosomal (aSTR) and Y-chromosomal Short Tandem 78 
Repeat (Y-STR) markers in order to compare the utility of these samples for human remains 79 
identification. 80 
 81 
2. Materials and Methods 82 
2.1. DNA sources 83 
Human remains from twelve (12) male persons that can be classified into two groups were used in 84 
the present study. The first group of human remains consisted of five (5) unidentified cadavers 85 
who were recently deceased wherein samples were collected within two (2) to nine (9) days post-86 
mortem. These cadavers were sent to the Department of Anatomy, College of Medicine, 87 
University of the Philippines, Manila (UPM-CM-DA) immediately after death, and stored at 88 
ambient temperature. The second group consisted of seven (7) male embalmed human remains 89 
that were exhumed ~1 month after internment by the Commission on Human Rights (CHR). 90 
Three (3) cadavers were buried inside individual coffins and interred in above-ground concrete 91 
vaults whereas four (4) cadavers were inside individual coffins that were buried below ground. 92 
 93 
Six types of bone samples namely femur, rib, clavicle, vertebra, patella, and metatarsal, were 94 
collected from the two groups of human remains. When available, vitreous fluid (VF), bone 95 
marrow from femur and clavicle, and muscle tissues were collected from the two groups of male 96 
remains. The state of one recently deceased cadaver did not allow for the collection of VF. A 97 
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vertebral sample was also not collected from one cadaver that was interred above ground for ~ 1 98 
month due to constraints encountered on site by our collaborating agency (CHR). Blood was 99 
collected from the five recently deceased cadavers in order to generate reference DNA profiles. 100 
 101 
This study was approved by University of the Philippines Manila, Research and Ethics Board 102 
(2012-0275-P1). 103 
 104 
2.2. Sample processing 105 
Heat-stable materials and equipment parts used were decontaminated via autoclaving at 121
o
C, 15 106 
lb/inch
2
 for 15 minutes, whereas heat-labile materials were UV-irradiated for at least 2 hours prior 107 
to use. 108 
 109 
Prior to processing, all samples were stored as follows: bone marrow and VF that were transferred 110 
on FTA
®
 cards (Whatman Intl., NJ), were stored at room temperature; blood and vitreous fluid 111 
samples collected with or without preservative (EDTA) were stored at 4
o
C; and bone, bone 112 
marrow and muscle tissue samples were stored at -20
o
C. Blood, VF and bone marrow on FTA
®
 113 
cards were processed after 1-7 days following manufacturer’s instruction. Bone samples were 114 
processed following a methodology reported previously [1]. Briefly, each bone sample was de-115 
fleshed then air-dried. Dried samples were sanded to remove external contaminants and were cut 116 
into 1-2 cm bone fragments using a rotary tool equipped with a new sanding band and cutting 117 
disc. Bone cuttings, weighing ~2.0 g, were washed twice in 5%Terg-a-zyme and sonicated for 25 118 
minutes. Bone cuttings were washed with sterile distilled water thrice. Additional washes with 119 
sterile distilled water were done until no more bubble was observed. Bone samples were dried in 120 
the oven at 56°C for 18-24 hours. After drying, bone cuttings were pulverized into fine powder 121 
using the Spex CertiPrep 6750 Freezer/Mill Cryogenic Grinder (SPEX SamplePrep LLC, NJ). 122 
Muscle tissue samples that were stored at -20
o
C, were brought to ambient temperature. Tissue 123 
samples were then minced into very fine portions using sterile disposable blades. The following 124 
were prepared for DNA extraction: five replicates of 0.1 g bone powder, bone marrow and muscle 125 
tissue samples; two replicates of 200 µl VF samples that were stored with or without EDTA. 126 
 127 
2.3. Organic DNA extraction with Microcon YM-100 Concentrators 128 
A one (1) ml lysis buffer solution consisting of 790 µL Tris-EDTA-NaCl (10 mmol/L Tris, pH 129 
8.0 – 50 mmol/L EDTA, pH 8.0 – 100 mmol/L NaCl) buffer, 100 µL SDS (20%), 40 µL DTT 130 
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(1.0 M) and 70 µL Proteinase K (20 mg/mL), was added to 0.1 g samples, incubated at 56°C for 131 
~24 hours with constant agitation set at 1,200 rpm in a Thermomixer
®
 comfort (Eppendorf, 132 
Hamburg). After incubation, an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) 133 
was added to the lysate. The lysate-PCI preparation was mixed, transferred to a High Density 134 
Phase-Lock Gel tube (Qiagen, Hamburg) and centrifuged for two minutes at 14,000 rpm to 135 
facilitate the separation of the aqueous and organic layers. The upper aqueous layer was then 136 
placed in Microcon YM-100 centrifugal filter units (Millipore, MA), washed once with TE
-4
 137 
buffer and eluted with 40µl TE
-4
 buffer. 138 
 139 
A similar procedure was used to extract DNA from VF, with slight modifications. 300 µL lysis 140 
buffer was added to 200 µL VF, incubated at 56°C for ~17 hours with constant agitation set at 141 
1,200 rpm. 500 µL PCI was then added to the lysate and extracted accordingly. 142 
 143 
2.4. DNA quantitation 144 
Determination of total human and Y-DNA concentrations and detection of presence of inhibitors 145 
from DNA extracts were conducted using the Plexor HY human DNAquantitation kit, reactions 146 
were ran in an AB 7500 Real-time PCR system (AB Life Technologies, CA) and analyzed using 147 
Plexor HY software v2 (Promega Corporation, WI). DNA concentrations (ng/μl) were multiplied 148 
with the elution volume (μL) and divided by the original amount or volume of sample in order to 149 
calculate DNA yield (ng) per initial weight of sample (g) or volume of fluid samples (μL). 150 
 151 
2.5. Autosomal STR (aSTR) and Y-chromosomal STR (YSTR) DNA profiling 152 
Amplifications of aSTRs and Y-STRs were performed using PowerPlex
®
21 (PP21) and 153 
PowerPlex
®
Y 23 (PPY23) (Promega Corporation), respectively, in reduced PCR reaction 154 
volumes. Two DNA template masses (0.1 ng and 0.5 ng) of bone and non-bone sample types, 155 
when applicable, per 12.5 μL reaction volume were used to compare the utility of different 156 
sample types as source of DNA for genotyping. Undiluted DNA extracts were amplified when 157 
estimated DNA concentration of a preparation is very low. Amplifications were done in a PE 158 
9700 thermocycler (AB Life Technologies) following manufacturer's instructions. Amplified 159 
products were separated via capillary electrophoresis and detected in an ABI PRISM 3500 160 
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Genetic Analyzer using GeneMapper
®
 ID-X v.1.2 software (AB Life Technologies), according to 161 
manufacturer’s recommended protocols. Allele designations were determined by comparison of 162 
the amplified DNA fragments with the allelic ladders supplied in the respective kits. 163 
 164 
The following parameters were evaluated to measure PCR success: 1) DNA yield; 2) peak height 165 
(PH) values in relative fluorescent units (RFU); 3) total allele recovery expressed as percentage of 166 
alleles generated compared to total expected alleles observed across all aSTR markers, with 167 
minimum peak height set at 50 rfu,; and 4) peak height ratio (PHR) in heterozygous alleles, 168 
defined as the smaller peak/larger peak. In untreated recently deceased samples, reference 169 
genotypes obtained from blood samples were compared with the DNA profiles that were 170 
generated using different sample types. In the absence of a reference sample and DNA profile in 171 
embalmed/exhumed remains, a consensus DNA profile was used as the reference DNA profile for 172 
each cadaver. 173 
 174 
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3. Results and Discussion 175 
3.1. DNA yield 176 
The average DNA yield of each sample type for untreated/unburied and embalmed/exhumed 177 
human remains (Figure 1). 178 
 179 
Figure 1. DNA yield of samples obtained from (A) untreated/unburied (B) embalmed/buried above ground and (C) 180 
embalmed/buried below ground human remains. 181 
 182 
Overall, sufficient amounts of DNA were recovered from bone samples even without a 183 
decalcification step. The removal of this step in the preparation of bone samples for DNA 184 
extraction, streamlines the entire bone processing procedures. Results of real-time assays showed 185 
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sufficient bone and non-bone DNA for subsequent genotyping were extracted from 0.1 g samples 186 
using organic based DNA extraction procedures. On the other hand, DNA extraction was not 187 
successful using vitreous fluid (VF) samples from embalmed/exhumed remains. It is possible that 188 
the fluid samples that were recovered from embalmed remains were mostly formalin. In contrast, 189 
vitreous fluid with or without EDTA from recently deceased cadavers that were not embalmed 190 
yielded varying amounts of DNA. Hence, vitreous fluid from untreated bodies may be a suitable 191 
source of DNA for human remains identification. Compared to bone and tissue samples, VF 192 
samples are much easier to collect, transport and process in a mass disaster scenario. 193 
 194 
DNA yield >1 ng/0.1g sample was obtained in 95% of the extracts. Generally, a lower DNA yield 195 
was observed in samples obtained from three human remains that were interred above ground. 196 
The presence of inhibitors was more consistently detected in samples (bone marrow, muscle 197 
tissue, rib and vertebra) from human remains with below ground burial. Detection of inhibitors 198 
may be related to soil contaminants and degradation products due to microbial activity that were 199 
not completely removed even after organic (PCI) purification. 200 
 201 
3.2. Allele recovery 202 
Complete DNA profiles were generated with 0.1 ng DNA template in 12.5 µL PCR reaction 203 
volume. Increasing DNA template to 0.5 ng improved allele recovery by 10-50% (Figure 2a-c). 204 
Amplifiable DNA with interpretable profiles was recovered in a majority of the samples analyzed 205 
even after formalin treatment and buried for ~ 1 month (Fig 2b-c). Our results show that formalin 206 
treatment in human remains has limited effect on the recovery and amplification of DNA in bone 207 
and muscle tissue samples. This is evidenced by a DNA yield >1ng per 0.1g original sample in 208 
majority of the extracts and the generation of complete STR DNA profiles. Partial profiles are 209 
likely due to insufficient target DNA (amplicons up to ~500 bp) amplified rather than DNA 210 
concentration. 211 
 212 
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Allele Recovery (%) 213 
A: untreated/unburied human remains 214 
 215 
 216 
 217 
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B: embalmed/buried above ground 218 
 219 
 220 
 221 
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C: embalmed/buried below ground 222 
 223 
 224 
 225 
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Figure 2. Autosomal and Y-STR alleles recovered (%) for each sample type. Comparison made between samples 226 
obtained from human remains that are (A) untreated/unburied (B) embalmed/buried above ground (C) embalmed/ 227 
buried below ground using two DNA template mass (0.1 and 0.5 ng) and on FTA in 12.5 µL reaction. 228 
 229 
Amplifiable target DNA (amplicons up to ~500 bp) suitable for aSTR and Y-STR DNA analysis 230 
was recovered from 0.1 g sample. Our data suggest that adequate quantities of DNA can be 231 
recovered from 0.1 g bone and non-bone material. Results of this study provide further empirical 232 
data supporting previous observations [12] that a small amount of bone starting material may be 233 
used for nuclear typing and appeared to performed better during amplification due to reduction in 234 
inhibition problems. Hence, small bone samples such as metatarsal and patella are also good 235 
DNA sources and should be collected during autopsy. 236 
 237 
DNA extracts from post mortem samples collected from untreated recently deceased cadavers 238 
showed similar DNA typing results regardless of the sample type use. For more challenged 239 
samples such as those collected from human remains that were embalmed and buried, data 240 
showed consistently higher allele recoveries from femur and bone marrow obtained from femur. 241 
In addition, complete to nearly complete profiles (>60%) were obtained from smaller and easier 242 
to collect bone types such as patella and metatarsal bones. 243 
 244 
Routine genotyping procedures have preferentially used compact bones as starting material for 245 
human remains identification. It was previously reported [11] that general trends in success rates 246 
of DNA analyses were observed with respect to the type of bone tested with the highest success 247 
rates observed with samples from dense cortical weight bearing leg bones, particularly with femur 248 
(86.9%), followed by teeth samples, with the lowest success rate from clavicle, ulna and radius. 249 
Bones that performed less tend to be less dense and/or have a greater proportion of spongy diploic 250 
bone. Here we have shown thatamplifiable nuclear DNA can be recovered consistently from 251 
femur and bone marrow samples. Depending on the condition of the samples, complete profiles 252 
can also be generated from foot and patella bone. Thus, these bone sample types could be used as 253 
an alternative DNA source when analyzing human remains. This is especially useful if the 254 
collection of femur bones is impractical due to the invasiveness of the procedure and the difficulty 255 
of obtaining these samples from a highly rigid, intact embalmed body. We succeeded in obtaining 256 
good quality DNA profiles from small and less compact bone samples such as patella and 257 
metatarsal (foot bone), similar to earlier reports [7, 13, 14]. There was an observed difference in 258 
allele recoveries dependent on the burial condition of the cadavers. Hence, the variability 259 
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observed warrants further investigation if this bone type is to be used as a standard routine sample 260 
for disaster victim identification. 261 
 262 
Results also show that amplifiable DNA can be generated from bone marrow taken from femur 263 
samples and transferred on FTA
®
 card which are cost efficient and easier to process. Complete to 264 
nearly complete (>80%) genetic profiles were recovered from bone marrow samples on FTA
®
 265 
from recently deceased/ unburied and those that were embalmed / buried below-ground with 266 
relatively balanced peaks for both aSTR and Y-STR DNA typing systems (Figure 3) whereas, no 267 
profile was observed for bone marrow samples buried above-ground. 268 
 269 
Bone marrow (femur) extracted via organic extraction method 270 
(A) Autosomal STR 271 
 272 
(B) Y-STR 273 
 274 
 275 
Bone marrow (femur) on FTA
®
 card 276 
(C) Autosomal STR 277 
 278 
 279 
 280 
 281 
 282 
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(D) Y-STR 283 
 284 
Figure 3. Representative electropherograms showing aSTR (A, C) and Y-STR (B, D) profiles from bone marrow 285 
(femur) samples obtained from human remains buried below ground extracted via organic procedure and using FTA
®
 286 
technology. All expected alleles were generated with relatively balance peak signals. 287 
 288 
Variation in allele recoveries for above ground vs. below ground bone marrow samples on FTA
®
 289 
can be related to the consistency of bone marrow and efficiency of transfer of samples onto the 290 
FTA
®
 card. Bone marrow samples taken from human remains in above-ground concrete vaults 291 
were drier and more difficult to transfer onto FTA
®
 cards, hence sample transfer was inefficient. 292 
 293 
3.3. Peak Height and Peak height Ratio of Heterozygous Alleles 294 
From post mortem samples included in this report, we efficiently amplified DNA extracts 295 
utilizing ~0.1 ng or greater initial DNA template load using PP21 and PPY23 multiplex system. 296 
Davoren and co-workers [15] reported successful amplification in bone DNA extracts generating 297 
complete aSTR profile with PowerPlex

16 using ≥150 pg DNA, whereas in another report, 298 
amplification of 100 pg or less DNA reproducible results with anticipated stochastic effects using 299 
the AmpFISTR Identifiler system [16]. Loss of heterozygosity and locus dropouts were most 300 
observed using 0.1 ng of starting DNA template. Increasing DNA template to 0.5 ng in 12.5 µL 301 
PCR reaction volume improved peak signals. Majority of scatter plot points clustered in the right 302 
quadrant indicating relatively balanced peaks in heterozygote alleles using 0.5 ng DNA template 303 
mass (Figure 4). With reduce template quantity in the PCR reaction (≤ 0.1 ng), stochastic effects 304 
such as allelic dropout and drop-in, intra and interlocus imbalance and poor spectral resolution i.e. 305 
stutter and bleedthroughs are some of the problems often encountered [16, 17, 18]. Although 306 
each replicate is equally likely to exhibit random artifacts, it is favorable to do multiple 307 
amplifications to clarify stochastic effects brought about by low template quantity and report 308 
consensus alleles generated from replicate analysis as recommended [16,17]. 309 
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 310 
Figure 4. Peak height (major peak) and heterozygote balance. Scatter plots of the minor peak to major peak ratio (in 311 
RFUs) using 0.1 ng and 0.5 ng DNA template. Specific loci evaluated include only those markers for which at least 312 
one of the two expected alleles was present in a given DNA profile. For allele dropouts, the PHR = 0. 313 
 314 
4. Conclusion 315 
We were able to recover and amplify DNA from bone samples taken from recently deceased 316 
untreated cadavers and embalmed/exhumed human remains using methods described here. 317 
Complete DNA profiles were generated from femur, bone marrow from femur, metatarsal and 318 
patella. If available, bone marrow and vitreous fluid samples can be used for genotyping, which is 319 
more cost-efficient for mass disaster identification efforts. The data also indicates that smaller 320 
bones such as patella and metatarsal (foot bone) which are thought to be poor DNA sources 321 
before can be utilized as alternative sources of DNA for STR typing. Amplifiable target DNA 322 
maybe obtained using 0.1 ng of DNA, increasing DNA template to 0.5 ng in 12.5 µL PCR 323 
reaction volume significantly improves allele recovery to up to 50% more, with interpretable and 324 
relatively balanced inter-locus peak signals. These results have clear implications in the 325 
identification of disaster victims, in situations where bone samples are used as DNA source. 326 
 327 
 328 
Acknowledgements 329 
The project was supported by the Philippine Council for Health Research and Development-330 
Department of Science and Technology (PCHRD-DOST) (FP:120044), the Philippine Genome 331 
Center and the Natural Sciences Research Institute, University of the Philippines, Diliman. We 332 
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thank Miriam Ruth M. Dalet, Minerva S. Sagum, Maria Lourdes D. Honrado and Paul Ryan L. 333 
Sales for their excellent technical support and Frederick C. Delfin for his valuable insights during 334 
the preparation of the manuscript. The authors acknowledged Kelly Baroga (UP-College of 335 
Medicine-Department of Anatomy) and the Commission on Human Rights-Forensic Center staff 336 
for their assistance during sample collection. 337 
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Disclaimers 401 
None 402 
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Conflict(s) of interest 404 
None 405 
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