Keywords age - PCR - pulp - dentin
Introduction
Age is one of the key parameters in establishing a physical characteristic profile
of an individual. For biological evidence left in crime scenes such as blood, saliva,
hair, etc., the evidence of owner's age can be determined only by DNA extracted from
these materials. Previous researchers have found that there are certain DNA regions
with specialized characteristic and function called telomere being able to predict
age.[1 ] Estimating tooth age and skeletal age are the two primary methods in age estimation
of forensic medicine. But they are often impacted with geographical environment, nutrition,
habitation, and ethnologic differences, so the accuracy will be reduced, especially
for the adult. With the study of telomere, it is certain that the length of the telomere
DNA can reflect the cell division and represent the cell lifespan, and it has some
pertinence to the age of the donor, so to measure the length of telomere DNA is a
new and valuable method for age estimation in the forensic medicine.[2 ]
Drastic change is seen in the criminal scenario, road traffic/rail/aviation accidents,
mass disasters, wars, and the bodies which are found are beyond recognition. When
segments of the body or cranial cavity or isolated teeth are found, sex identification
becomes the most intriguing, complex, and sometimes controversial challenge. Teeth
being the hardest substances in the human body, potentially can survive most of the
insults and consequences encountered at death and during decomposition. Tooth pulp
remains protected in a hard tissue casing made up of dentin and enamel. So, the present
study is planned to be conducted on pulp tissue and dental hard tissues derived from
individuals for DNA isolation and age determination.[3 ]
The aim of the present study is to assess the viability of human dental pulp as a
source for DNA and to establish a reproducible, simple, and standardized protocol
for age determination by polymerase chain reaction (PCR) method.
We adopted O'Callaghan et al's modified method of absolute quantification of telomere
length by introducing oligomer standard to generate telomere values.[4 ]
Materials and Methods
Study sample was calculated by using a formula by using random sampling technique
and 30 extracted teeth of known gender and age were taken. The study included tooth
samples of known age range from 18 to 40 years. Tooth samples immediately after extraction
were taken, in which vital tooth was considered before extraction, but not vital pulp.
Permanent tooth samples extracted for orthodontic treatment, impacted teeth or due
to periodontal destruction. There were few exclusions from the study such as tooth
samples whose age was not known. The study was done exclusively on permanent tooth.
Deciduous tooth samples were excluded from the study. As it was a new study, we wanted
more quantity of pulp tissues rather than necrotic pulp so teeth with extensive decay
involving pulp were excluded. Tooth with wear facets were excluded because most of
the time pulp chamber and canal would be calcified in the presence of ware facets
restored and RC treated teeth.
The present study was an experimental single-blinded study consisting of 30 extracted
teeth categorized into three groups as follows Group A: 10 to 20 years, Group B: 21
to 30 years, Group C: 31 to 40 years. Two investigators were involved in the extraction
of pulp and dentine in the study. Initially teeth were washed in 5.2% sodium hypochlorite
solution for around 30 seconds, and then the teeth were cleaned and washed again with
sterile distilled water for around 30 seconds. The tooth was wiped with cotton and
by using hand trimmer with carborundum disk; each tooth was longitudinally sectioned
into two halves. The pulp tissue from each tooth was removed by using an endodontic
broach no. 21. The derived pulp sample was then put in a sterile Eppendorf tube containing
a Tris EDTA buffer (10-mM Tris buffer, 1 mM EDTA, pH 7.5). The tubes were labeled
accordingly and stored at normal room temperature.
Samples of pulp were then sent to the Department of Molecular Biology and Immunology
to the second observer who did not know the age of the patient, for further procedure.
In the next step, DNA extraction procedure (Modified Proteinase-K method) for pulp
was done.[2 ] Briefly, the samples were vortexed and then washed with fresh TE buffer three times.
This is followed by the addition of lysis buffer containing 10-mm Tris buffer and
1-mm EDTA and Lysis buffer II containing 50-mm Tris HCl, 50-mm KCL, MgCl2 2.5 mm, Tween 20 0.45%, and nonidet-P 0.45%. Proteinase K (10 mg/mL) was added to
degrade the protein contaminants and kept at 60°C in water bath for 2 hours followed
by enzyme deactivation by keeping in boiling water bath for 10 minutes. The DNA was
collected from the supernatant after centrifugation at 5,000 rpm for 5 minutes.
Quantitative PCR (qPCR) for calculating telomere length was performed as described
by O'Callaghan et al. Standard curve is used to calculate absolute telomere length.
A standard curve is set up by the dilution of known quantities of a synthesized 84
mer oligonucleotide containing only TTAGGG repeats ([Table 1 ]). The amount of telomere sequence in TEL STD is calculated as 1 × 108 kb of telomere sequence in TEL STD as determined by using Avogadro's number. A single
copy gene (SCG, 36B4) is used as a control for amplification of every sample performed
and to determine genome copies per sample. The genome copy per reaction is calculated
as 1 × 109 diploid genome copies. A serial dilution of TEL STD was performed (1.0 × 108 to 1.0 × 104 kb telomere sequences). A serial dilution of SCG STD was also performed simultaneously
(1 × 109 to 1 × 105 dilution).
Table 1
Oligomers used in the study
Sequence name
Oligomer sequence (5 to3′)
Standards
Telomere standard
(TTAGGG)14
36B4 standard
CAGCAAGTGGGAAGGTGTAATCCGTCTCCACAGACAAGGCCAGGACTCGTTTGTACCCGTTGATGATAGAATGGG
PCR primers
teloF
CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT
teloR
GGCTTGCCTTACCCTTACCCTTACCC TTACCCTTACCCT
36B4F
CAGCAAGTGGGAAGGTGTAATCC
36B4R
CCCATTCTATCATCAACGGGTACAA
Source: Adapted from O'Callaghan N et al.[4 ]
Telomere for each sample including TEL STD was amplified by using telomere-specific
primers (TeloF and TeloR) by qPCR to get kb/reaction of telomere for each sample.
Single copy gene (36B4) for each sample including SCG STD was amplified using SCG-specific
primers (36B4F and 36B4R) by qPCR to get diploid genome copy number for each sample.
CT (cycle threshold) is the cycle number at which fluorescence signal is generated.
Telomere standard curve was generated by plotting CT values against amount of telomere
sequence in kb per reaction ([Fig. 1A ]). SCG standard curve was generated by plotting CT values against 36B4 genome copies
([Fig. 1B ]). The standard curves and graphs were generated by using Realplex software (Eppendorf,
Hamburg, Germany). the values of telomere kb per reaction and diploid copy number
of each sample were exported into excel format. The telomere kb per reaction value
was divided by diploid genome copy number to give a total telomeric length in kb per
human diploid genome.
Fig. 1 Standard curve used to calculate absolute telomere length. CT (cycle threshold) is
the cycle number at which fluorescence signal is generated. (A ) Graph showing standard curve for calculating length of telomere sequence per reaction
tube. Slope −3.4 and efficiency is 0.96. (B ) Graph showing standard curve for calculating genome copies using 36B4 copy number.
Slope −3.4 and efficiency is 0.95. Standard curves were generated using an Realplex
software (Eppendorf, Hamburg, Germany).
Results
Age determination from tooth pulp was planned and determined through real time (qPCR)
in the present study. Group A comprised of 09 samples with an age range of 11 to 20
years, which showed a varied telomere length between 9.57 to 11.05, and a mean telomere
length was 9.92 ([Table 2 ]). Group B comprised of 17 samples with an age range of 21 to 30 years, which showed
a varied telomere length between 8.64 and 10.26, and a mean telomere length was 9.3
([Table 3 ]). Group C comprised of only four samples with an age range of 31 to 40 years, which
showed a varied telomere length between 7.32 and 10.25, and a mean telomere length
of 9.1275 ([Table 4 ]). When the telomere length was compared between the groups, the mean telomere length
was found to be decreasing from Group A to Group C. Telomere length was decreased
in Group B when compared with Group A and similarly in Group C when compared with
Group A and Group B. With the results of present study, we have assessed that, as
the age increases the length of telomere gets shortened which will be helpful in analyzing
the age of the person when morphological and biological remnants are not available
except tooth.
Table 2
Group A: 11 to 20 years
Sample no.
Actual age
Telomere length
1
15
9.57
2
15
9.06
3
15
10.04
4
15
9.57
5
18
10.06
6
19
10.09
7
19
10.09
8
20
11.05
9
20
9.88
Mean telomere length
9.92
Table 3
Group B: 21–30 years
Sample no.
Actual age
Telomere length
1
21
10.08
2
21
8.64
3
22
10.06
4
23
10.24
5
23
10.12
6
23
10.14
7
23
10.24
8
24
10.26
9
24
9.83
10
24
10.12
11
24
10.26
12
24
9.83
13
24
9.05
14
26
10.14
15
26
10.25
16
29
9.89
17
29
10.25
Mean telomere length
9.3
Table 4
Group C: 30–40 y
Sample no
Actual age
Telomere length
1
32
7.32
2
32
9.89
3
35
9.05
4
35
10.25
Mean telomere length
9.1275
Discussion
There are many methods of estimation of age that are based on anatomical changes such
as seen in ribs, hands, and teeth. There are no really good methods for age estimation
within the forensic area. A sample of evidence usually carries no morphological information
as for instance, a blood stain. In these cases, a method based on molecular observations
would be preferred. It has been shown that human telomeres shorten with time.[5 ] The anatomical position of the dental pulp protects it from various stimuli such
as temperature, microbes, or oral fluid. Pulp offers the best source of DNA for reliable
genetic analysis in forensic science. DNA is an identical unit of each individual.
The smallest amount of DNA can divulge and decipher the biggest mystery. The quality
and quantity of pulp tissue will depend on the environmental insult.[6 ] This is the first study to access the age by telomere length using dental pulp.
In the present study, we were able to retrieve pulp tissue in all the groups which
were considered. Pulp which was retrieved was sufficient to amplify the DNA in the
study. Naik et al[7 ] was able to retrieve the mean DNA quantity of 26.41 ng/μL. In our study, extracted
teeth showed a wide range of DNA quantity. Reason for the same was the varying size
of the pulp cavity, which is directly proportional to the age of the participant.
Similar to the present study Khan et al,[8 ] stated in her study that among the total of 20 samples, DNA isolation was done from
all the samples of pulp and dentin. Similarly, Battepati and Shodan,[9 ] in their study achieved 100% positive results in amplifying the DNA quantity extracted
from 30 teeth buried in the soil for 2 months.
In our study we showed that the variation in the telomere repeats is considerably
observed in dentine pulp tissues. There is no doubt that the telomeres shorten through
life, but the degree of this loss varied among different individuals as shown. There
will be variations in telomere length in various tissues. What is the cause of the
variation seen between humans? Some minor variation might be due to the method itself.
However, biological factors such as diseases and lifestyle as well as inherited telomere
length and telomerase activity most probably affect the actual telomere length. Several
human diseases, in addition to aging in general, are known to be linked to telomeres.
In cancer the rate of cell division affects the rate of telomere length. The environment
of the cell is also a factor that affects the telomere length external factors such
as the level of oxidative stress and the efficiency of the antioxidant defense. Stress
and cigarette smoking have been shown to lead to a higher degree of oxidative stress
not only these up-regulation of the immune system (i.e., infection) could have an
impact on the result.[10 ]
Conclusion
The present study found that estimation of human age based on the relative TL measured
by the real-time quantitative PCR may be a useful method for age prediction, especially
when there is no morphologic information in the biological sample. Meanwhile it could
only give a rough estimation of age or could be assigned to an age interval. To exactly
analyze and determine the age, real-time quantitative PCR assay is simple, rapid,
and readily scalable to achieve a high throughput of samples. To exactly analyze the
age group of the patient between the age group more number of samples needs to be
taken with equal number of samples in each group.
