Dental Forensic Kit (DFK®) and Quick Extract™ FFPE DNA extraction kit, a new workflow for obtaining dental DNA for human genetic identity

Abstract

Background
Most of the protocols described for obtaining DNA from dental tissues are methods that involve major laboratory equipment and many hours of work. They are also methodologies that almost destroy the tooth.

Aim
Our aim was to develop an innovative workflow for optimizing dental DNA extraction from teeth. Our methodological proposal is a new workflow for obtaining dental DNA for human genetic identity using Dental Forensic Kit (DFK®) and Formalin-Fixed Paraffin-Embedded (FFPE™) DNA extraction kit.

Methods
Two different dental samples groups were assayed with DFK® and FFPE™. The first group corresponded to extracted teeth from living donors and the second group was considered in real conditions with challenging teeth from corpses. Genomic Dental DNA was amplified and genotyped with platforms Identifiler Plus™, Power Plex 21™ and Global Filer ™ kits.

Results
Our workflow was useful in obtaining dental DNA and partial and complete genetic profiles, from teeth of both study groups. DFK® kit worked in a conservative treatment of teeth generating dental tissues (pulp and cement) and FFPE™ for DNA extraction was a very cheap, quick and easy method for obtaining genomic dental DNA.

Conclusions
The innovative method and the workflow proposed herein allows obtaining robust and reliable genetic profiles, from dental tissues. DFK® kit works optimizing the treatment of dental tissues and FFPE™ demonstrates a new use in forensic genetics.

Full text

Journal of Forensic and Legal Medicine 73 (2020) 101992
Available online 2 June 2020
1752-928X/© 2020 Published by Elsevier Ltd.
Research Paper
Dental Forensic Kit (DFK®) and Quick Extract™ FFPE DNA extraction kit, a
new workow for obtaining dental DNA for human genetic identity
Carolina Inostroza
a
,
*
, Patricio Carrasco
a
, Marianela Godoy
a
, Gianna Gatti
b
, Beatriz Paulino
a
a
Universidad de Los Andes. Dental School, Monse~
nor 
Alvaro del Portillo 12.455, Las Condes, Santiago, PO7620001, Chile
b
Servicio M
edico Legal de Chile, Avenida La Paz 1.012, Independencia, Santiago, PO8380454, Chile
ARTICLE INFO
Keywords:
Genetic prole
Human identity
Forensic dentistry
ABSTRACT
Background: Most of the protocols described for obtaining DNA from dental tissues are methods that involve
major laboratory equipment and many hours of work. They are also methodologies that almost destroy the tooth.
Aim: Our aim was to develop an innovative workow for optimizing dental DNA extraction from teeth. Our
methodological proposal is a new workow for obtaining dental DNA for human genetic identity using Dental
Forensic Kit (DFK®) and Formalin-Fixed Parafn-Embedded (FFPE™) DNA extraction kit.
Methods: Two different dental samples groups were assayed with DFK® and FFPE™. The rst group corre-
sponded to extracted teeth from living donors and the second group was considered in real conditions with
challenging teeth from corpses. Genomic Dental DNA was amplied and genotyped with platforms Identiler
Plus™, Power Plex 21™ and Global Filer ™ kits.
Results: Our workow was useful in obtaining dental DNA and partial and complete genetic proles, from teeth of
both study groups. DFK® kit worked in a conservative treatment of teeth generating dental tissues (pulp and
cement) and FFPE™ for DNA extraction was a very cheap, quick and easy method for obtaining genomic dental
DNA.
Conclusions: The innovative method and the workow proposed herein allows obtaining robust and reliable
genetic proles, from dental tissues. DFK® kit works optimizing the treatment of dental tissues and FFPE™
demonstrates a new use in forensic genetics.
1. Introduction
Different methods have been explored to obtain and analyze bio-
logical material from victims exposed to adverse conditions-such as vi-
olent impacts and explosions from plane crashes and terrorist acts, and
natural disasters-often been exposed to long time and an accelerated
decomposition of the body. In such cases, tissues available for forensic
analysis are often limited to bone and/or teeth.
1,2
Nowadays, obtaining
genomic DNA (gDNA) with a high degree of purity and integrity for the
genetic identication of this type of human remains is still challenging.
The oral cavity, a rich reservoir for cells and body uids, is known to
resist and preserve its components, especially teeth, from external in-
uences.
3
These solid structures within the mouth are rich in gDNA- and
mitochondrial DNA (mtDNA)- containing cells, both in soft and hard
tissues. Respectively, DNA is found internally, in the dental pulp tissue
(4), and on the external surface of the dental root in cementocites and
broblasts of periodontal tissues(5).
Different approaches are used to access dental tissue and obtain
DNA, such as grinding and cryogenic pulverization,
6–9
the inclusion of
teeth in a solid matrix,
10,11
and endodontic access with the use of
rotatory instruments.
8,12,13
Some of their common characteristics are
the decontamination of teeth with sodium hypochlorite. This phase
meant to clean the surface has proven to deteriorate the cellular layer of
root cement and destroy soft tissue. Different types of decontamination
processes and improvement of de-hydrated tissues as a pre-treatment
would enable the recovery of tissue and gDNA with higher integrity
for better analysis with forensic kits and platforms.
5
Important limitations lie in the length of protocols and time required
for genetic typing. Both relate to excessive handling of the sample ma-
terial, which increases contamination risks(14). Ultimate partial or total
destruction of the evidence obstructs further physical or biochemical
analysis (such as determining time or cause of death, toxicology, and
anthropological studies) and handing the remains over to relatives.
15,16
Cryogenic pulverization is the predominant approach for gDNA
* Corresponding author.
E-mail address: caroviriffo@gmail.com (C. Inostroza).
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine
journal homepage: http://www.elsevier.com/locate/yjflm
https://doi.org/10.1016/j.jm.2020.101992
Received 8 January 2020; Received in revised form 18 May 2020; Accepted 23 May 2020

Journal of Forensic and Legal Medicine 73 (2020) 101992
2
extraction from hard tissue in humans(9,15,17). However, it requires
the use of expensive equipment and a strictly controlled environment
from the recovery of the biological material to the generation of genetic
proles. Also, they require long processing steps, there is a high risk for
contamination, and at the same time powdering of calcied structures
favors PCR inhibition.
18–24
It depends on specialized staff for the use and
maintenance of equipment, while only a few samples can be worked in
parallel. Moreover, a large amount of sample is required for the pro-
cedure, which is then destroyed and unable to be reanalyzed.
15,16
Endodontic access is the less destructive methodology, however,
methodological aspects are very heterogeneous between different
studies(6,8,12, 13, 24–26).
Natural degradation processes could inuence the availability of
gDNA from teeth. Boy et al. studied dental pulp degradation using ow
cytometry for DNA and found that there was a minimal DNA degrada-
tion of dental pulp tissue even after 144 h post-extraction, and also that
this degradation had no linear pattern(27). Studies that focus on the
histologic changes after death have found that cellular components of
dental pulp degenerate progressively, together with the migration of
cells to the periphery(28–32). Young et al. found differential degrada-
tion of RNA β-actin amplicons of dental pulp until 84 days of PMI(33). In
1989, Yamada and Yamamoto(34) reported the rst gDNA prole ob-
tained from dental pulp, while other studies have determined root
cement and periodontal tissues as another rich source of DNA.
5,25,35
Therefore, dental tissues are recognized as an excellent source for high
amounts of DNA which is better conserved than in bone in highly
degraded samples.
3–5
In this research, we propose an alternative, less destructive workow
for obtaining samples from dental remains for human genetic identity:
Dental Forensic Kit (DFK®) and Quick Extract™ FFPE DNA extraction
kit. The Dental Forensic Kit (DFK®) was specically designed to opti-
mize dental tissue retrieval in routine forensic nuclear DNA and/or
mitochondrial DNA genotyping and forensic histopathology applica-
tions.
32
Our methodology submits teeth to pre-treatment simulating
physiological-like conditions (such as humidity, 37 �C, salinity, pro-
teoglycan (hyaluronic acid) similar to lymph that ows on live tissues)
generation a process of re-hydration that contributes to the effective
obtainment of viable genetic material from tooth samples,
32,39
which
permits the obtainment of the dental pulp and root cement (two distinct
biological tissues from the same tooth) quickly and efciently. As a
result, high-quality gDNA is extracted for subsequent genetic proling,
while we physically preserve the tooth. The suggested protocol
combining forensic odontology with forensic genetics can be done in a
short amount of time, an important target in human identity testing.
38
2. Materials and methods
Our research followed the ethical guidelines and approval of the
Ethics Research and Compliance Committee of the Dental School from
the University of Los Andes.
2.1. Preparation procedures, teeth samples, DNA extraction, and DNA
analysis
2.1.1. Teeth samples selection
In the workow, we obtained a non-random set of teeth. Donors who
agreed to donate their teeth signed an informed consent (Online
resource) specially designed for the present research. 11 teeth were
collected from donors of both genders (7 men and 4 women) at the
Dental Clinical Service Cedin Ltda. at Paine City in Chile.
The inclusion criteria for teeth were extractions for orthodontic
reasons and the exclusion criteria implied the presence of caries, llings,
crowns or veneers, fractures, and endodontic treatment. Teeth were
extracted by the same calibrated operator. On the same day of the tooth
extraction, teeth collected under sterile conditions were placed directly
into individually labeled sterile tubes and transferred to the laboratory
and kept at room temperature between 18 and 25 �C, atmospheric
pressure (1 bar) and 40% humidity until the experimentation with
DFK®. The PMI in this study was considered as the lapse between loss of
vitality (date of extraction) and experimental analysis. The PMI of teeth
ranged from seven days up to 18 years.
2.1.2. DFK® assay
Samples were treated under the instructions of the DFK® kit, pre-
viously described by Carrasco et al. (US 61/826, 558 May 23, 2013
Patent)
37
(Fig. 1). The teeth were treated under conditions designed to
minimize contamination (samples were kept in sterile tubes, all pro-
cedures were done on the laminar ow chamber, all materials and so-
lutions were properly sterile before use, and operators wore gloves,
masks and face protectors and hairnets during all the experiments). Each
tooth was kept on a 5 mL polypropylene sterile tube (Mic Biologic
Rubilator, S.L., and Barcelona). Digital radiography was taken for each
sample using Sirona, Heliodent, and Charlotte (NC 2827 Successive,
USA) equipment, with measurements analyzed with the SIDEXIS soft-
ware (Sirona, Gensheim, Germany) to accurately plan access to the
pulpar chamber. Each sample was then washed on a saline solution
buffered at pH 7.4 by phosphate (Phosphate Buffered Saline, PBS 1x,
Hyclone) with 3 successive washes on a volume of 10 mL during 1 min
and vortexed (Vortex Mixer Mrc). Then, samples were immersed in the
Fig. 1. Critical steps of the methodology and kit DFK.
C. Inostroza et al.

Journal of Forensic and Legal Medicine 73 (2020) 101992
3
external hydration solution from DFK® (PBS 1x supplemented with
hyaluronic acid and salts) in an electrophoresis chamber for 1 h at 300
mA, following immersion during 12 h at 37 �C in the same solution on a
culture oven. In a vertical ow hood, each tooth was placed on a petri
dish, ready for pulp chamber perforation with a turbine with diamond
circular burs of 1–2 mm diameter, according to the distances measured
upon radiography. Teeth were perforated on the occlusal surface until
communication with the pulp chamber and on the apical third of the
root to access the root canal. Subsequently, each sample was immersed
in a sterile internal hydration solution from the DFK® (Stem cell culture
media without antibiotics and fetal bovine serum) and immersed for 48
h at 37 �C and 5% CO
2
in an incubator MCO 17 AC Sanyo 1. Once hy-
drated, samples were taken into the vertical ood hood. The dental pulp
was removed with Automated Endodontic File (1.5/25 mm (SAF; Redent
Nova, Ra’anana, Israel), and root cement was sliced from the radicular
surface with a nº15 sterile scalpel and deposited on a petri dish. After the
removal of the pulp and cement dental tissues, teeth perforations were
sealed. For restoration we performed UV-light induced polymerization,
using CharmBOND® and CharmFil® Plus (Dentkist Inc.) with a dental
curing light LED.D (Guilin Woodpecker Woodpecker Medical Instru-
ment Co., Ltd.). Tissues were subsequently washed with sterile DNA-free
water in preparation for DNA extraction. Trepanation, tissue removal,
and washes were carried out under negative pressure in a vertical ow
hood. Laboratory operators had been trained in the correct use of the
DFK®.
2.1.3. DNA extraction and quantitation
Root cement (RC) and dental pulp (P) tissues from teeth assayed with
the DFK®. All samples were treated with a quick (1 h) one step Quick
Extract™ FFPE DNA Extraction Kit (Epicenter®) according to supplier
instructions.
Total gDNA from root cement and dental pulp were quantied in a
NanoDrop™ 2000/2000c (Thermo Scientic™) or with the Kit Quan-
tiler® Duo DNA Quantication (Life Technologies, Carlsbad, CA; USA,
Carlsbad, CA, USA.) in a Real-Time PCR 7500 (Life Technologies,
Carlsbad, CA; USA) (Tables 1 and 2).
DNA Samples 5LD, 6LD, 7LD, and 8LD were assayed in an external
laboratory (Crime Investigation Laboratory of the Chilean Police (PDI))
using Maxwell® 16 System, Promega Corporation, the most common
automated gold standard kit in forensic laboratories.
DNA preparation for the Polymerase Chain Reaction (PCR) was
carried out according to the Kit Quantiler ® Duo DNA Quantication
(Life Technologies, Carlsbad, CA; USA, Carlsbad, CA, USA) User’s
Manual provided, in a Real-Time PCR System, Applied Biosystems 7500.
2.1.4. DNA analysis
DNA Genotyping was done using capillary electrophoresis (CE). The
kit GlobalFiler™ PCR Amplication kit (Life Technologies, Carlsbad,
CA; USA) was used for genotyping DNA from samples 1LD, 2LD, 3LD,
and 4LD. AmpFLSTR® Identiler® Plus PCR amplication kit (Life
Technologies, Carlsbad, CA; USA) was used for genotyping DNA from
samples 9LD, 10LD, and 11LD. POWER PLEX 21® system (Promega™)
was used for samples 5LD, 6LD, 7LD, and 8LD.
For pulp from 5LD and root cement from samples 6LD and 11LD,
DNA extraction was unsuccessful; therefore, genotyping was not done in
these samples.
Electrophoresis was run on a 3500 Genetic Analyzer (Life Technol-
ogies, Carlsbad, CA; USA™). Alleles were designated according to the
recommendations of the DNA Commission of the International Society
of Forensic Genetics (ISFG) using the software Gene Mapper® IDX v1.4
(Life Technologies, Carlsbad, CA; USA). All resulting genetic proles
obtained from the teeth samples were compared to those of laboratory
personnel using Gene Mapper ID-X software version 1.0 (Life Technol-
ogies, Carlsbad, CA; USA, Applied Biosystems, Foster City, CA, USA). All
procedures were performed according to supplier instructions and
considering internal kit validation.
2.2. Proof of concept DFK® workow
For proof of concept DFK®, we collected 5 teeth from 4 corpses with
a PMI of 1 month. We established that teeth fullled the following
criteria: healthy teeth with no caries, llings, and fractures. Teeth were
extracted by the same calibrated operator. On the same day of the tooth
extraction, teeth collected under sterile conditions were placed directly
into individually labeled sterile tubes and were transferred to the lab-
oratory and kept at room temperature between 18 and 25 �C, atmo-
spheric pressure (1 bar) and 40% humidity until the experimentation
with DFK®. The corpses belonged to male individuals between 47 and
Table 1
Sample characteristics, DNA quantication and STRs score of teeth from living donors.
Code
a
Gender
b
Age in years PMI
c
Tooth type
d
NanoDrop DNA quantication
μ
g/
μ
l
qPCR DNA quantication
μ
g/
μ
l
h
STR score/16, 21, 24
STRs
e
Amelogenin
f
RC P RC P RC P RC P
1LD M 54 1 M 10 D C NA NA 0,075 46,2 24/24 24/24 XY XY
2LD F 18 7 D TM NA NA 2,14 134 22/24 22/24 XX XX
3LD M 46 13 D TM NA NA 0,013 0025 -/24
g
24/24 XY XY
4LD M 51 2 M PM NA NA 0,113 87 24/24 24/24 XY XY
5LD M 25 15 D PM NA NA 0,087 0001 16/16 – XY –
6LD M 48 18 Y PM NA NA NT 0,028 – 13/21 – XY
7LD M 43 1 Y 3 M LI NA 0,0002 0,284 NA 21/21 21/21 XY XY
8LD M 43 1 Y 3 M CI NA NA 1,14 0,34 20/21 21/21 XY XY
9LD F 23 6 M TM 0,01 0,010 NA NA 13/16 16/16 X X-
10LD F 20 9 M TM 0,01 0,016 NA NA 13/16 16/16 X XX
11LD F 23 2 Y 2 M TM NT 0,00045 NA NA – 3/16 – XX
RC ¼root cement; P ¼dental pulp.
NA ¼not applied; NT ¼no tissue obtained; Indet ¼Indeterminate results, out of range3.
a
Code: LD: Living donor.
b
Gender: M ¼male, F ¼female.
c
PMI: Postmortem interval; Y ¼years, M ¼months; D ¼days.
d
Tooth type: C: canine; LI: lateral incisor; CI: central incisor; PM: premolar; TM: third molar.
e
STR score/16, 21, 24STR: STR score obtained of a total of STRs 16 (Identiler plus™), 21 (Power Plex 21®) and 24 (Global Filer™). Genotyping of the control of
DNA extraction CT did not show STR amplication.
f
Amelogenin: XY ¼male; XX ¼female; - ¼no STR results or no tissue obtained.
g
Contamination of electropherogram.
h
Control of DNA extraction was measured by Q-PCR showed 0,007
μ
g/
μ
l.
C. Inostroza et al.

Journal of Forensic and Legal Medicine 73 (2020) 101992
4
83 years old. Blood samples in FTA®cards were obtained as reference
genetic proles.
All corpses samples (1C, 2C.1, 2C.2, 3C, and 4C) were assayed with
the DFK® workow and then treated with one step Quick Extract™
FFPE DNA Extraction Kit (Epicenter®) according to supplier’s in-
structions. Total gDNA from root cement and dental pulp were quanti-
ed in a NanoDrop™ 2000/2000c (Thermo Scientic™) and also with
the Kit Quantiler ® Duo DNA Quantication (Life Technologies,
Carlsbad, CA; USA, Carlsbad, CA, USA) in a Real Time PCR 7500 (Life
Technologies, Carlsbad, CA; USA)(Table 2).
Subsequently genotyping was done using capillary electrophoresis
for both root cement and pulp DNAs, if available. We used GlobalFiler™
PCR Amplication kit (Life Technologies, Carlsbad, CA; USA™) for
samples 1C, 2C.1, 2C.2, 3C and 4C. In sample 3C there was no pulp tissue
available for the DNA extraction and genotyping, but DNA from root
cement was successfully genotyped. Electrophoresis was run on a 3500
Genetic Analyzer (Life Technologies, Carlsbad, CA; USA). Alleles were
designated according to the recommendations of the DNA Commission
of the International Society of Forensic Genetics (ISFG) using the soft-
ware Gene Mapper® IDX v1.4 (Life Technologies, Carlsbad, CA; USA).
All resulting genetic proles obtained from the teeth DNA samples were
compared to those of two laboratories operators using Gene Mapper ID-
X software version 1.0 (Applied Biosystems™ 3500 Thermo Scientic™,
Foster City, CA, USA). All procedures were performed according to
supplier instructions and considering internal kit validation. Genetic
proles from operators are shown in Supplemental Material.
The blood collected in FTA® cards were genotyped with direct
amplication Buffer, Prep-n-go™ (Applied Biosystems™ 3500 Thermo
Scientic™) followed by GlobalFilerExpress™ kit (Life Technologies,
Carlsbad, CA; USA™) in Real Time Gen Amp 9700 PCR (Applied Bio-
systems). Samples were injected in a 3500 Genetic Analyzer (Life
Technologies, Carlsbad, CA; USA) for mapping with the Gene Mapper®
IDX v1.4 software (Life Technologies, Carlsbad, CA; USA). Genetic
proles from samples of group 2 cadavers are shown in Supplemental
Material.
For classication of genetic proles we used the reference study re-
ported by Amory et al. and Holland et al.
36,38
: A full prole informed on
all loci (16 for Identiler Plus™, 21 for POWER PLEX® and 24 for
GlobalFiler™), an acceptable prole for 11/16, 16/21 and 21/24 loci,
quite acceptable for between 8 and 10 loci and an unsuitable prole
informed on less than 8 loci.
2.3. Statistical analysis
Descriptive and association statistical analysis was done for samples
(pulp and root cement independently) using genetic prole classica-
tion as a dependent variable and independent variables were teeth PMI,
teeth type, age of donor, and gender of the donor. Due to the small
sample size, Fisher’s exact test and Wilcoxon rank-sum test were applied
with the software STATA 13.0 with a signicant p value of 0,05.
Software Familias 3.0
39
was applied for genotype comparisons be-
tween pulp and root cement samples and between proof of concept
samples and blood references. Matching probabilities of genotypes using
the blind search module was done. Results were expressed in Likelihood
ratios. Allele frequencies for the 15 STRs used (CSFIPO, D13S317,
D16S539, D18S51, D19S433, D21S11, D2S1338, D3S1358, D5S818,
D7S820, D8S1179, FGA, THO1, TPOX, VWA) corresponded to the
reference frequency database of Chile published in 2015(40).
3. Results
Living donors (LD) were 7 men and 4 women, aged between 18 and
54 years. The PMI of teeth extracted ranged from seven days to 18 years.
After DNA extraction, we determined DNA concentration either with
Nanodrop or qPCR showed in Table 1. For samples measured with
Nanodrop, concentration values ranged between 0,026 and 0,016
μ
g/
μ
l,
while for those samples measured with qPCR, we observed values be-
tween 0,001–134
μ
g/
μ
l. For genotyping, we used different platforms
according to laboratory availability. Four pulp samples and four root
cement samples were genotyped with GlobalFiler™ for 24 STRs. Five
samples of this group got complete genetic proles (1LD: P and RC, 3LD:
P, 4LD: P and RC), two samples got partial genetic proles with 22 STR
allele calls (2LD: P and RC) and only one sample did not get a genetic
prole (3LD: RC). Three pulp and two root cement samples were gen-
otyped with POWERPLEX® for 21 STRs. Of these, three samples showed
complete genetic proles (7LD: P and RC, 8LD: P) (Fig. 2) and two
samples got partial genetic proles with 13–20 allele calls (8LD: RC and
6LD: P). Three pulp and three root cement samples were genotyped with
Identiler Plus™ for 16 STRs. Of these, three samples had a complete
prole (5LD: RC, 9LD: P, 10LD: P) and three samples had partial proles
(9LD: RC, 10LD: RC and 11LD: P) from 3 to 13 STR allele calls. No
statistically signicant associations were observed concerning the age of
the donor, PMI of teeth, the gender of donor or tooth type concerning
full prole, acceptable prole, quite acceptable or unsuitable prole (p
>0,05). Direct match probabilities of all samples (dental pup and root
cement) were calculated as Likelihood ratios (LR). All samples from the
same teeth showed high LR values (Table 3), whereas samples from
different teeth showed negative LR’s (data not shown); meaning there is
a high probability that dental pulp and root cement are from the same
individual. Samples from tooth 7LD and 8LD showed high LR between
them, presuming that both teeth are from the same person.
Dental DNA extraction was done with the Quick Extract™ FFPE DNA
extraction kit according to the supplier’s instructions described in the
methods section. DNA concentrations were determined with Nanodrop
or qPCR, data showed in Table 2. For samples measured with Nanodrop
values ranged between 0,0068 and 0,253
μ
g/
μ
l, while for those samples
measured with qPCR, values were between 0,0012–45
μ
g/
μ
l.
According to the proof of concept, corpses samples 1C, 2C.1, 2C.2,
Table 2
Sample characteristics, DNA quantication and STRs score of teeth from cadavers.
Gender
a
Age in years PMI
b
Tooth type
c
NanoDrop DNA quantication
μ
g/
μ
l qPCR DNA quantication
μ
g/
μ
l
f
STR score/24 STRs
d
Amelogenin
e
RC P RC P RC P RC P
1C M 54 1 M C 0,168 0253 0,45 Indet 10/24 24/24 XY XY
2C.1 M 47 1 M PM 0,60 0,020 45 2,29 24/24 24/24 XY XY
2C.2 M 47 1 M C 0,84 0,104 25,87 0,04 24/24 24/24 XY XY
3C M 63 1 M LI Indet NT 5,1 NT 13/24 – – –
4C M 83 1 M PM 0,94 0,0068 0,0012 0,004 23/24 17/24 XY XY
RC ¼root cement, P ¼dental pulp; NA ¼not applied; NT ¼no tissue obtained; Indet ¼Indeterminate results, out of range.
a
Gender: M ¼male.
b
PMI: Post mortem interval; , M ¼month.
c
Tooth type: C ¼canine; PM ¼premolar; LI ¼lateral incisor.
d
STR score 24STR: STR score obtained of a total of STRs 24 (Global Filer™).
e
Amelogenin: XY ¼male; - ¼no STR results or not tissue obtained.
f
Control of DNA extraction was measured by Q-PCR showed 0,007
μ
g/
μ
l.
C. Inostroza et al.

Journal of Forensic and Legal Medicine 73 (2020) 101992
5
3C, and 4C were genotyped for 24 STRs. Pulp and root cement from
samples 2C.1 and 2C.2 got a complete genetic prole and Pulp from 3C
and pulp and root cement from 4C produced a partial genetic prole.
Likelihood ratios (LR) were calculated to evaluate the matching proba-
bilities between pulp and root cement with the corresponding blood
sample from the same corpse to determine genetic identity. Values are
shown in Table 4. Only sample 3C showed no correspondence between
RC and blood (B) reference genotype.
None of the genetic proles obtained from sampling materials
matched the proles of operators involved in the process.
4. Discussion
Our study aimed to show the genetic results associated with the
quick proposal DFK® for obtaining samples from dental remains for
human genetic identity. Our interest was focused in dental DNA from
both tissues: dental pulps and root cements. The DFK® methodology
could be considered as a less destructive method, since it does not
destroy the tooth during its experimentation. The main difference be-
tween DFK® and other less destructive methodologies
6,8,10–13,25,26
is the
presentation of pretreatment of the tooth before tissue retrieval. Our
methodology submits teeth to pre-treatment simulating
physiological-like conditions (such as humidity, 37 �C, salinity, pro-
teoglycan (hyaluronic acid) similar to lymph that ows on live tissues)
generation a process of re-hydration that contributes to the effective
obtainment of viable genetic material from tooth samples,
32,39
which
permits the obtainment of the dental pulp and root cement (two distinct
biological tissues from the same tooth) quickly and efciently.
32,37
Fig. 2. Electropherograms from dental pulp and root cement sample 7LD, 21STRs of 21STRs genotyped with Power Plex 21.
Table 3
Likelihood ratios for direct match probability between Pulp and root cement
STRs from teeth of living donors. Direct match probabilities of all samples
(dental pup and root cement) were calculated as Likelihood ratios (LR). All
samples from the same teeth showed high LR values, whereas samples from
different teeth showed negative LR’s (data not shown); meaning there is a high
probability that dental pulp and root cement are from the same individual. In
fact, samples from tooth 7LD and 8LD showed high LR between them, presuming
that both teeth are from the same person.
DENTAL PULP SAMPLE ROOT CEMENT SAMPLE LR
1LD P 1LD RC 3,Eþ21
2LD P 2LD RC 3,Eþ22
4LD P 4LD RC 1,Eþ22
7LD P 7LD RC 5,Eþ25
7LD P 8LD P 5,Eþ25
7LD P 8LD RC 2,Eþ19
7LD RC 8LD P 5,Eþ25
7LD RC 8LD RC 2,Eþ19
8LD P 8LD RC 2,Eþ19
9LD P 9LD RC 8,Eþ20
10LD P 10LD RC 2,Eþ27
P¼Pulp.
RC ¼Root cement.
LR ¼likelihood ratio.
Table 4
Likelihood ratios for direct match probability between teeth and blood STRs
from cadavers.
DENTAL PULP SAMPLE ROOT CEMENT SAMPLE
LR
2C.2 P 2C B 2,Eþ20
2C.2 RC 2C B 2,Eþ20
2C.1 P 2C B 2,Eþ20
2C.1 RC 2C B 3,Eþ17
3C RC 3C B 2,E04
4C P 4C B 1,Eþ22
4C RC 4C B 7,Eþ03
P¼Pulp.
RC ¼Root cement.
B¼Blood.
LR ¼likelihood ratio.
C. Inostroza et al.

Journal of Forensic and Legal Medicine 73 (2020) 101992
6
DFK® maximizes the potential of the tooth for DNA recovery from in-
ternal and external tissues: dental pulp and root cement, respectively. In
the workow, we considered the use of QuickExtract™ FFPE DNA
Extraction Kit (Epicenter® and Illumina Company), commonly used in
parafn-embedded tissues, while looking for a quick, easy and cheap
extraction method for gDNA extraction. Despite its few user references
with no proven efcacy in generating full genetic proles in forensic
casework samples, we realized it is possible to obtain useful gDNA for
STR genotyping (Tables 1 and 2). Our results conrm that this DNA
extractor reduces laboratory time, is equipment-effective compared to
other automated systems such as Maxwell® 16 System, Promega Cor-
poration [33–35], FastPrep 24™ -5G (MP Biomedicals) [36]), manual
(ChargeSwitch® Forensic DNA Purication Kit [16] and QIAamp® DNA
Mini Kit by Qiagen™ [37,38]) methods (based on manufacturer infor-
mation). Therefore, we consider that QuickExtract™ FFPE DNA
Extraction Kit is an effective method for forensic DNA analysis in this
proposed workow.
According to the DNA quantication analysis, we did not nd any
associations in the DNA concentration values between Nanodrop and
qPCR results. The Nanodrop has the disadvantage that it does not
differentiate human from non-human DNA, generating high yields of
DNA. On the other hand, qPCR results varied between DNA concentra-
tions values very low, undetermined, or out of range. On this matter, we
hypothesize that the QuickExtract™ reagents may interfere in the
uorescence method of quantication by Real-Time PCR. CT/IPC values
showed the optimum CT value ranged from 28 to 31 for samples 1C,
2C.1, and 2C.2 in the dental pulp and root cement. But this hypothesis
must be tested. Only in samples 3C and 4C we found higher CT values,
indicative of the presence of PCR inhibitors. Invalid IPC value was
observed in 3C, suggesting DNA degradation or nucleic acids absence
(Table 2).Samples from living donors got 11 complete genetic proles (7
from DNA in pulp and 4 DNA in root cement), 6 produced acceptable
genetic proles (4 DNAs from root cement and 2 pulp) and 2 unsuitable
from root cement and dental pulp respectively, both with contamination
mixture with more than one allelic assignment. Because we are aware
that genotyping was done under different methods, is that we decided to
analyze prole results in terms of its utility to inform identity as reported
by Amory et al. and Holland et al.(36,38).
Likelihood ratios for direct match probabilities between pulp and
root cement DNA samples from the same donors are shown in Table 3.
Samples 3LD, 5LD, 6LD, and 11LD were excluded from analysis because
either pulp or root cement DNA yield no results (Table 1). Positive and
high LR values for samples from the same teeth, and unexpectedly we
determined that teeth 7LD and 8LD came from the same person
(Table 3). Even though the matching probabilities were expected to be
positive between samples from the same donor, the results are an
example of the utility of the present methodology, showing that though
a partial or unsuitable prole may be obtained with one of the sampled
tissues, there is the possibility for better success in regards to the other
tissue, and also no contamination was is seen between the samples. No
signicant statistical association was found between pulp genetic pro-
les and PMI of the tooth, type of tooth, age or gender of the donors (p >
0,05); nor between root cement genetic proles and PMI of the tooth,
type of tooth, age or gender of the donors or the genotype prole (p >
0,05). The absence of association may be due to the small number of
samples available. However, the results suggest that higher PMIs
generate lower STR scores. Still, the genetic prole with a score of 13/21
STRs obtained for sample 6LD, with a PMI of 18 years, is highly valuable
in forensic DNA analysis. We thus determined its utility for being used in
forensic contexts, including probably mass disasters.
1,2
For sample
recruiting, we established that teeth fullled the following criteria:
healthy teeth with no caries, llings, and fractures. Since 60% of the
proles were complete we decided to apply the same criteria to the proof
of concept sampling.
DFK® methodology was applied real conditions samples as a proof of
concept. 4 samples showed a complete genetic prole with 24/24 STRs
(2C.1 and 2C.2: P and RC) and 2 samples showed an acceptable prole
with 13–23-17/24 respectively (3C: RC and 4C: P and RC). From sam-
ples 2C.1 RC and P, and 2C.2 P the LR of matching identity against blood
reference data was 2
Eþ20
and 2C.2 RC was 3
Eþ17
because of an allelic
dropout. The LR for samples 4C P and 4C RC against blood were 1
Eþ22
and 7
Eþ03
, respectively. Those differences were seen because both
samples showed partial proles (23–17/24), nevertheless, both were
informative and allowed identity recognition. The only sample that did
not show concordance with the blood sample was 3C (LR ¼2
E04
) due to
possible contamination or degradation of gDNA. Samples from 1C were
not included in statistical analysis because of artifacts on the genotype.
Allelic assignment on GenMapper against reference proles generated
from blood reference samples together with genetic statistics conrmed
the efciency of the proposed methodology (Table 4).
There are some limitations regarding the quality of genotypes. It was
observed a saturated pattern of electropherograms, and this might be
due to the high concentration values from qPCR, which was reected in
DNA input. We strongly suggest that in the presence of enough DNA,
samples should be re-run for better accuracy in the results. For cases
where genetic typing of gDNA failed to produce a full or acceptable
prole, we suggest a step of DNA extraction using purication methods
or kits in an attempt to improve DNA yield. Finally, the less destructive
aspect of this methodology permits leaving the tooth practically intact
after an adequate sealing. The damage resulting from the aggressive
steps described in previous methodologies
9,15,16,41,42
(such as cryogenic
pulverization and cutting) can be avoided, thus the sample is used for
further analysis and/or returned to the relatives of the
victim.
6–9,13,14,22,23,26,33
In this scenario, DFK has the utility for being
used in forensic and historical/anthropological contexts, including mass
disasters.
1,2
To complete the total workow – until the generation of genotypes-
takes approximately 26,8 h (Fig. 1) in comparison to the estimated time
involved in classical methods for bones and teeth, described previously
in the introduction(9,15,17–21,23–25,36,44). Moreover, several sam-
ples can be prepared and worked on simultaneously (except for the
perforation steps). For this reason, we consider this methodology to be
cost-effective concerning others and also may be performed by any
forensic specialist after proper instruction. On the other hand, we did not
aim to perform an analysis regarding costs at the time of this research,
since further studies must be performed with more samples. From our
results, we hypothesize that methodologies that contemplate destruc-
tion of the tooth should be overcome in forensic analysis. We
acknowledge such protocols have been longed used in forensic odon-
tology and genetics. However, we support that the present workow has
the potential to improve the efciency and utility of the sample material,
thus improving forensic DNA testing. The possibilities of yielding results
in forensic samples, which are known to be challenging and often
degraded could be increased by the possibility of generating two distinct
dental tissues that might be available from a single sample. In this case,
we demonstrated that DNAs from dental pulp and root cement are good
candidates for identity genetic analysis. In our study, no specic type of
teeth was associated with the performance of genotyping, but we can
suggest choosing caries-, lling- and fracture-free teeth for working with
the DFK workow. We also recommend using root cement as the rst
option for analysis, because the root cement removal is easier to do
although there is a higher risk for PCR inhibition in root cement from
calcium and collagen(21) compared to the dental pulp.
This workow is an alternative for the specialists in forensic labo-
ratories that need to solve challenging cases.
45
A challenge for the future
is to evaluate the workow in a larger sample size with a greater number
of cases in different environmental conditions.
5. Conclusions
Dental Forensic Kit (DFK®) and Quick Extract™ FFPE DNA extrac-
tion kit, are presented as a new useful workow for obtaining dental
C. Inostroza et al.

Journal of Forensic and Legal Medicine 73 (2020) 101992
7
DNA for human genetic identity. The DFK® is time-effective and reliable
while maximizing the informative value of the sample material for
forensic investigation purposes. As a support methodology, the Quick
Extract™ FFPE DNA Extraction kit proved to be easy and quick in
generating good quality DNA.
Declaration of competing interest
The authors declare that they have no conict of interests.
CRediT authorship contribution statement
Carolina Inostroza: Supervision, Investigation, Formal analysis,
Writing - original draft. Patricio Carrasco: Conceptualization, Formal
analysis, Writing - review & editing. Marianela Godoy: Data curation,
Methodology, Validation. Gianna Gatti: Funding acquisition, Re-
sources. Beatriz Paulino: Project administration, Software.
References
1. Tan P, Wee KP, Sahelangi P. Remembering the musi-SilkAir ight MI 185 crash
victim identication. Ann Acad Med Singapore. 2007;36(10):861.
2. Gaudry E, Dourel L, Conigliaro A, Georget C, Desbrosse X, Hubac S. L’identication
de victimes de catastrophe: une approche scientique pluridisciplinaire. Revue
Francophone des Laboratoires. 2017;2017(489):41–57.
3. Diwaker NR, Rajeshwari null, Rao B. DNA ngerprinting. The future of forensic
dentistry–a review. Indian J Dent Res. junio de. 2001;12(2):81–88.
4. Malaver PC, Yunis JJ. Different dental tissues as source of DNA for human
identication in forensic cases. Croat Med J. 2003;44(3):306–309.
5. Higgins D, Austin JJ. Teeth as a source of DNA for forensic identication of human
remains: a Review. Science & Justice. diciembre de. 2013;53(4):433–441.
6. Smith BC, Fisher DL, Weedn VW, Warnock GR, Holland MM. A systematic approach
to the sampling of dental DNA. J Forensic Sci Soc. 1993;38(5):1194–1209.
7. Sweet D, Hildebrand D. Recovery of DNA from human teeth by cryogenic grinding.
J Forensic Sci. noviembre de. 1998;43(6):1199–1202.
8. Tilotta F, Brousseau P, Lepareur E, Yasukawa K, de Mazancourt P. A comparative
study of two methods of dental pulp extraction for genetic ngerprinting. Forensic
Sci Int. 10 de octubre de. 2010;202(1-3):e39–43.
9. Sivagami AV, Rao AR, Varshney U. A simple and cost-effective method for preparing
DNA from the hard tooth tissue, and its use in polymerase chain reaction
amplication of amelogenin gene segment for sex determination in an Indian
population. Forensic Sci Int. 15 de mayo de. 2000;110(2):107–115.
10. Tran-Hung L, Tran-Thi N, Aboudharam G, Raoult D, Drancourt M. A new method to
extract dental pulp DNA: application to universal detection of bacteria. PloS One.
2007;2(10), e1062.
11. Gilbert MTP, Willerslev E, Hansen AJ, et al. Distribution patterns of postmortem
damage in human mitochondrial DNA. Am J Hum Genet. enero de. 2003;72(1):32–47.
12. Carrasco P, Espinoza N, Mery A. Obtaining of DNA from Dental Pulps with Forensic
Purposes. Use of AMP-FLP Technique Specic for APO-B Locus. Dentistry faculty,
University of Chile; 1993.
13. Pinchi V, Torricelli F, Nutini AL, Conti M, Iozzi S, Norelli G-A. Techniques of dental
DNA extraction: some operative experiences. Forensic Sci Int. 30 de enero de. 2011;
204(1-3):111–114.
14. Thomas P, Gilbert M, Rudbeck L, et al. Biochemical and physical correlates of DNA
contamination in archaeological human bones and teeth excavated at Matera, Italy.
Journal of Archaeological Science. mayo de. 2005;32(5):785–793.
15. Loreille OM, Diegoli TM, Irwin JA, Coble MD, Parsons TJ. High efciency DNA
extraction from bone by total demineralization. Forensic Science International:
Genetics. junio de. 2007;1(2):191–195.
16. K€
ochl S, Niederst€
atter H, Parson W. DNA extraction and quantitation of forensic
samples using the phenol-chloroform method and real-time PCR. Methods Mol Biol.
2005;297:13–30.
17. Edson SM. Getting ahead: extraction of DNA from skeletonized cranial material and
teeth. J Forensic Sci. noviembre de. 2019;64(6):1646–1657.
18. Betancor a. An efcient method of extracting DNA from bone remains from the
Spanish civil war—a comparative study of two methods: PrepFiler BTA
TM
and
DNAzol® methods. Forensic news [Internet]. enero de 2011 [citado 26 de diciembre
de 2017]; Disponible en http://www.appliedbiosystems.com/ForensicNews-Past.
19. P€
a€
abo S. Ancient DNA: extraction, characterization, molecular cloning, and
enzymatic amplication. Proc. Natl. Acad. Sci. U.S.A. 1989;86(6):1939–1943.
20. Akane A, Shiono H, Matsubara K, Nakamura H, Hasegawa M, Kagawa M.
Purication of forensic specimens for the polymerase chain reaction (PCR) analysis.
Forensic Science International. mayo de. 1993;38(3):691–701.
21. McCord B, Pionzio A, Thompson R. Analysis of the Effect of a Variety of PCR Inhibitors
on the Amplication of DNA Using Real Time PCR, Melt Curves and STR Analysis. 2015.
22. H€
oss M, Kohn M, P€
a€
abo S. Excrement analysis by PCR. Nature. de septiembre de
1992;17:359.
23. Francalacci P. DNA recovery from ancient tissues: problems and perspectives.
Human Evolution. enero de. 1995;10(1):81–91.
24. Caero C, Re A, Stigliano E, Bassotti E, Moroni R, Grippaudo C. Optimization of
DNA extraction from dental remains. Electrophoresis. de mayo de 2019;29.
elps.201900142.
25. Damgaard PB, Margaryan A, Schroeder H, Orlando L, Willerslev E, Allentoft ME.
Improving access to endogenous DNA in ancient bones and teeth. Scientic Reports
[Internet], [citado 3 de noviembre de 2017];5(1). Disponible en http://www.nature.
com/articles/srep11184; septiembre de 2015.
26. Dutta P. Amelogenin gene - the pioneer in gender determination from forensic
dental samples. JOURNAL OF CLINICAL AND DIAGNOSTIC RESEARCH [Internet];
2017 [citado 7 de noviembre de 2017]; Disponible en http://jcdr.net/article_full
text.asp?issn¼0973-709x&year¼2017&volume¼11&issue¼2&page¼ZC56&issn¼0
973-709x&id¼9407.
27. Boy SC, Bernitz H, Van Heerden WFP. Flow cytometric evaluation of postmortem
pulp DNA degradation. Am J Forensic Med Pathol. junio de. 2003;24(2):123–127.
28. Gawande M, Chaudhary M, Das A. Estimation of the time of death by evaluating
histological changes in the pulp. Indian J Forensic Med Toxicol. 2012;6(2):80.
29. Mehendiratta M, Jain K, Boaz K, Bansal M, Manaktala N. Estimation of time elapsed
since the death from identication of morphological and histological time-related
changes in dental pulp: an observational study from porcine teeth. J Forensic Dent
Sci. 2015;7(2):95.
30. Caballín A, Per
ea B, De Agustín B, S
anchez JA. Valoraci
on de los cambios
histol
ogicos pulpares para la determinaci
on de la data de la muerte. Cient dent. 2010;
7(1):9–13.
31. Vavpotic M, Turk T, Martincic DS, Balazic J. Characteristics of the number of
odontoblasts in human dental pulp post-mortem. Forensic Sci Int. 15 de diciembre de.
2009;193(1-3):122–126.
32. Carrasco PA, Brizuela CI, Rodriguez IA, Mu~
noz S, Godoy ME, Inostroza C.
Histological transformations of the dental pulp as possible indicator of post mortem
interval: a pilot study. Forensic Science International. octubre de. 2017;279:251–257.
33. Young ST, Wells JD, Hobbs GR, Bishop CP. Estimating postmortem interval using
RNA degradation and morphological changes in tooth pulp. Forensic Sci Int. junio de
2013;229(1-3), 163.e1-163.e6.
34. Yamada Y, Yamamoto K, Yoshii T, Ishiyama I. Analysis of DNA from tooth and
application to forensic dental medicine. Nihon Hoigaku Zasshi. octubre de. 1989;43
(5):420–423.
35. Higgins D, Rohrlach AB, Kaidonis J, Townsend G, Austin JJ. Differential Nuclear and
Mitochondrial DNA Preservation in Post-Mortem Teeth with Implications for Forensic and
Ancient DNA Studies; 2015 [citado 27 de septiembre de 2015]; Disponible en
http://dx.plos.org/10.1371/journal.pone.0126935.
36. Amory S, Huel R, Bili
c A, Loreille O, Parsons TJ. Automatable full demineralization
DNA extraction procedure from degraded skeletal remains. Forensic Sci Int Genet.
mayo de. 2012;6(3):398–406.
37. Carrasco Tapia P, Inostroza Silva C. Inventors. Universidad de los Andes, asignee.
Method, systems and kit for forensic identication, post mortem interval estimation
and cause of death determination by recovery of dental tissue in physiological
conditions. United States patent US. 2013;61/826, 558 23.05.
38. Holland MM, Cave CA, Holland CA, Bille TW. Development of a quality, high
throughput DNA analysis procedure for skeletal samples to assist with the
identication of victims from the World Trade Center attacks. Croat Med J. junio de.
2003;44(3):264–272.
39. Kling D, Tillmar AO, Egeland T. Familias 3 – extensions and new functionality.
Forensic Sci Int: Genetics. 1 de noviembre de. 2014;13:121–127.
40. Toscanini U, Moreno F, Pantoja-Astudillo JA, Morales EA, Bustos P, Salas A.
A reference frequency database of 15 autosomal STRs in Chile. Forensic Sci Int:
Genetics. noviembre de. 2015;19:35–36.
41. Giuliani C, Cilli E, Bacalini MG, et al. Inferring chronological age from DNA
methylation patterns of human teeth: inferring age from DNA methylation OF
human teeth. American Journal of Physical Anthropology. abril de. 2016;159(4):
585–595.
42. Liu Q, Liu L, Zhang M, et al. A simple and efcient method of extracting DNA from
aged bones and teeth. Journal of Forensic Sciences [Internet]. 14 de diciembre de
2017. https://doi.org/10.1111/1556-4029.13603 [citado 27 de diciembre de
2017]; Disponible en.
44. Zupani
c Pajni
c I, Debska M, Gornjak Pogorelc B, et al. Highly efcient automated
extraction of DNA from old and contemporary skeletal remains. J Forensic Leg Med.
enero de. 2016;37:78–86.
45. Carrasco P, Inostroza C, Didier M, et al. Optimizing DNA recovery and forensic
typing of degraded blood and dental remains using a specialized extraction method,
comprehensive qPCR sample characterization, and massively parallel sequencing.
Int J Legal Med [Internet]; 14 de agosto de 2019 [citado 22 de noviembre de 2019];
Disponible en http://link.springer.com/10.1007/s00414-019-02124-y.
C. Inostroza et al.