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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 4  |  Page : 562-568
Sex determination from the pulp tissue of deciduous teeth exposed to natural soil and wet clay - A PCR study


NITTE (Deemed to be University), AB Shetty Memorial Institute of Dental Sciences, Department of Pediatric and Preventive Dentistry, Deralakatte, Mangalore, Karnataka, India

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Date of Submission30-Jan-2019
Date of Decision15-Feb-2019
Date of Acceptance28-May-2019
Date of Web Publication16-Oct-2020
 

   Abstract 


Context: Dental tissue remains are the toughest, and chemically, the most stable tissue in the body. Its high resilience in the events of fire and bacterial decomposition makes them vital for DNA analysis by PCR method. Aims: Determination of sex of children through molecular analysis of pulp tissue of exfoliated deciduous teeth stored in different media and analyzed after a different time period. Settings and Design: Sixty samples of deciduous teeth were divided into three groups. Group IA and Group IIA were stored in natural soil and wet clay for 1 month, respectively. Group IB and Group IIB were stored in natural soil and wet clay for 6 months, respectively. Group III was analyzed immediately after extraction. Methods and Material: Sex determination was carried out in five steps: Pulp tissue removal, DNA isolation, DNA quantification, PCR amplification, Sex determination. X and Y specific chromosomes from each sample were amplified and compared. Statistical Analysis Used: Kruskal-Wallis test, Dunn's test, and Wilcoxon signed rank test. Results: Group III revealed the highest amount of DNA quantified. Amount of DNA quantified after 6 months of storage in natural soil and wet clay decreased in both the groups with the samples stored in wet clay showing a maximum decrease. Results of the PCR analysis also showed 100% accuracy rate in the samples of Group III. Conclusions: Sex determination from pulp tissue depends a lot on the quality and quantity of DNA extracted. Sex could be effectively determined among the samples evaluated immediately after extraction. This ability decreases as the storage condition changes and the time period increases. Samples stored in wet clay were found to show the least sex identification ability than dry soil.

Keywords: Deciduous dentition, DNA isolation, DNA quantification, PCR analysis

How to cite this article:
Suman P, Manju R, Shetty VA, Hegde AM, Muthtamil, Rao S. Sex determination from the pulp tissue of deciduous teeth exposed to natural soil and wet clay - A PCR study. Indian J Dent Res 2020;31:562-8

How to cite this URL:
Suman P, Manju R, Shetty VA, Hegde AM, Muthtamil, Rao S. Sex determination from the pulp tissue of deciduous teeth exposed to natural soil and wet clay - A PCR study. Indian J Dent Res [serial online] 2020 [cited 2020 Oct 31];31:562-8. Available from: https://www.ijdr.in/text.asp?2020/31/4/562/298425



   Introduction Top


Forensic Odontology or Forensic Dentistry has been one of the most unexplored and fascinating divisions of forensic sciences.[1],[2]

Studies have proven over time that dental remains are excellent material in both existing and non-existing populations for odontologic, forensic, anthropological, and genetic investigations.[3],[4] Teeth are both toughest and chemically very stable tissue in the body. They are selectively conserved and fossilized, hence, providing for the paramount records for evolutionary change.[5],[6],[7] They even have high resilience in the events of fire and bacterial decomposition, hence, making them vital for deoxyribose nucleic acid (DNA) analysis.[8] The enamel and dentin of human teeth act like a shield to the DNA rich pulp tissue present beneath the dentin from various environmental insults.[9] Hence, it becomes a significant material for identification by confirmation of genetic markers.[10],[11],[12],[13]

With the development of gene analysis techniques like polymerase chain reaction (PCR) and sex determination using X and Y chromosomes have been conducted as a part of forensic investigation.[13],[14],[15] Although there are no steadfast methods of sex determination in deciduous dentition, PCR has proved to be extremely effective owing to its increased specificity, sensitivity, and speed.[16]

Another most important variable to be considered while determining sex in forensic investigation is the time period and type of medium the teeth were in contact with prior to the analysis. The availability of DNA depends largely on these factors as the type of soil, water, and room temperature have a significant effect on the dehydration rate of the pulpal tissue.[15]

Hence, owing to the above-mentioned facts, the present study was undertaken to find out the efficacy of sex determination in children from exfoliated deciduous teeth, immediately after extraction and stored under natural soil and wet clay for 1 month and 6 months, respectively by PCR.


   Materials and Methods Top


Sixty deciduous teeth were collected from children between the age group of 6–13 years who reported to the Department of Pedodontics and Preventive Dentistry for the extraction of their mobile deciduous teeth. Informed consent was obtained from the parents, and assent was obtained from the children. Ethical clearance was obtained from the Institutional Ethical committee.

Inclusion criteria

Samples from children of both sexes.

Exclusion criteria

Parents who have not given consent.

Sample size determination

To complete sample size, the technique of estimation of proportion (gender determination),

Sample size = [z1 – (α/2) 2 P (1–P)]/d2

α= level of significance (5%)

d = precision (15%)

P = anticipated proportion (80%)

Calculated sample size = 20, for each group. Therefore, for three groups = 60.

The teeth were divided into three groups of 20 samples each and stored in different environmental conditions for different periods of time before PCR analysis. Coding of all samples was done [Figure 1].
Figure 1: Division and coding of all samples in various groups

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The pulp tissue was removed from all the samples and collected using the sterile disposable needle and spoon excavator from the pulp chamber. The collected pulp tissue was stored in labeled bottles of Dulbecco's phosphate-buffer saline and transferred it to the research laboratory [Figure 2]a and [Figure 2]b.
Figure 2: (a and b) Collected pulp tissue in labeled bottles

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DNA isolation from the pulp tissue was done using the NucleoSpin Tissue kit (DSS Takara Bio India Pvt. Ltd). The tissue sample was placed in a microcentrifuge tube of 1.5 ml, and 180 μl of buffer T1 was added to 25 μl of proteinase K solution and vortexed [Figure 3]. Care was taken that the sample was completely covered with the lysis solution. The samples were then incubated at 56°C until complete lysis was obtained. Samples were then vortexed vigorously and incubated at 70°C for 10 min, and 600 μl of buffer was added and centrifuged at 11000 rpm for 1 min [Figure 4].[17]
Figure 3: Vortex mixer – was used to vortex the samples occasionally to lyse the samples and adjust the DNA binding conditions for DNA isolation

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Figure 4: Centrifuge – the tissue sample was placed in a microcentrifuge tube of 1.5 ml, and then, centrifuged to prepare the sample for DNA isolation

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The DNA isolated from the pulpal tissue of each sample was then quantified in μg using BioSpectrometer [Figure 5], to analyze the amount of DNA obtained from the samples of each of the Groups I, II, and III.[18],[19]
Figure 5: BioSpectrometer – for quantification of DNA samples in μg

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After the quantification of DNA, amplification of each DNA sample was done using PCR technique. In the present study, one Y chromosome-specific primer (size 172 bp) and one X chromosome-specific primer (size 131 bp) were used for PCR analysis [Figure 6]. Heat stable Taq DNA polymerase was provided in the PCR Buffer kit.[17],[20] The PCR products were then electrophoresed in 2% agarose gel at 80–100 V for 1 h [Figure 7], ethidium bromide staining was performed, and amplified bands of Y- and X- specific band sequences were examined under UV irradiation. The sex of the subjects was considered to be male when both Y- and X- specific sequences were detected, but female when only X-specific sequences were detected [Figure 8]a and b].[21],[22],[23]
Figure 6: X and Y- chromosome specific PCR primers used

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Figure 7: Gel electrophoresis- was used for the electrophoresis of PCR products in 2% agarose gel at 80–100V for 1 h

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Figure 8: (a) Detection of X and Y specific chromosomes (male samples). Both X and Y specific DNA bands amplified. M- DNA ladder, NC- Negative control, and bp- Base pairs. (b) Detection of X-specific chromosome (female sample). X- specific DNA band observed but Y specific DNA band not amplified. M-DNA ladder

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Descriptive and analytical statistics were done. The data did not follow a normal distribution; hence, non-parametric tests were used to analyze the data, as they are distribution-free tests and according to fewer assumptions. The normality of data was analyzed by the Shapiro-Wilk test. The Kruskal-Wallis test was used to check the mean differences among the groups. Posthoc analysis was done using Dunn's test. False discovery rate (FDR) controller was used for adjustments for applying Dunn's test. The Wilcoxon signed rank test was used to check the mean differences between the groups. The results of DNA quantification and PCR analysis of each group were statistically analyzed using SPSS (Statistical Package for Social Sciences) Version 24.0 (IBM Corporation, Chicago, USA).


   Results Top


The DNA quantification (μg) from tooth pulp and PCR analysis among the three groups was compared.

DNA quantification

Comparison of DNA quantification between group IA, group IIA, and group III

The DNA quantification (μg) of tooth pulp among the three groups was compared. Group III (immediately after tooth extraction) had the highest DNA quantification value (μg) (16.42 ± 6.63) followed by Group IA - tooth stored in natural soil for 1 month (5.66 ± 3.26) and group IIA – tooth stored in wet clay for 1 month (4.80 ± 3.26). The difference between the groups in mean DNA quantification (μg) was statistically significant (P < 0.001).

When Group IA was compared with Group IIA, a mean difference of 0.86 (95% C.I. –5.98–7.70) was found, which did not show any statistical significance (P = 0.638). When Group IA was compared with Group IIA, a mean difference of –12.07 (95% C.I. –18.91–5.22) was found, which was statistically significant (P < 0.001). When Group IIA was compared with Group III, a mean difference of –12.93 (95% C.I. –19.77–6.08) was found, which was statistically significant (P < 0.001) [Table 1], [Table 2] and [Graph 1].
Table 1: Comparison of DNA quantification(μg) of tooth pulp among the three groups: Group IA- tooth stored in natural soil for 1 month, Group IIA- tooth stored in wet clay for 1 month, and Group III – immediately after tooth extraction

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Table 2: Post hoc pair wise comparison of DNA quantification (μg) of tooth pulp among the three groups: Group IA- tooth stored in natural soil for 1 month, Group IIA- tooth stored in wet clay for 1 month, and Group III- immediately after tooth extraction

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Comparison of DNA quantification among Group IB, Group IIB, and Group III

The DNA quantification (μg) of tooth pulp among the three groups was compared. Group III (immediately after tooth extraction) had the highest DNA quantification value (μg) (16.42 ± 6.63) followed by Group IA - tooth stored in natural soil for 1 month (5.66 ± 3.26) and Group IIA – tooth stored in wet clay for 1 month (4.80 ± 3.26). The difference between the groups in mean DNA quantification (μg) was statistically significant (P < 0.001) [Table 3] and [Graph 2].
Table 3: Comparison of DNA quantification (μg) of tooth pulp among the three groups: Group IB. tooth stored in natural soil for 6 month, Group IIB- tooth stored in wet clay for 6 month, and Group III- immediately after tooth extraction

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PCR analysis

Intragroup comparison of the PCR analysis among Group IA and Group IB revealed 100% accuracy rate in Group IA with no false-positive or false-negative results. However, the accuracy rate of Group IB was determined as 80% with 50% false-positive results.

The intragroup comparison of PCR analysis of Group IIA and Group IIB was done. The samples of Group IIA showed 88.89% accuracy rate with 50% rate of false-positive results, which further declined to 66.67% accuracy rate in Group IIB and 33.33% false-positive results.

On comparing the results of the PCR analysis among Group IA and Group IIA, samples of Group IA (stored in natural soil for 1 month) showed 100% accuracy, specificity, and sensitivity rates. However, the accuracy rate dropped down to 88.89% in Group IIA (stored in wet clay for 1 month). The samples of Group IIA also recorded a low- specificity rate of 50% along with a false-positive rate of 50%.

On comparing the results of the PCR analysis among Group IB and Group IIB, samples of Group IB (stored in wet clay for 6 months) showed 80% accuracy rate, as compared to Group IIB, which showed a further drop in the accuracy rate of 66.67%. The rate of false-positive results in Group IB was 50% as compared to Group IIB, which was recorded as 33.33%. The sensitivity rate of Group IB was recorded as 100% with 66.67% recorded in Group IIB. The specificity among the groups was recorded as 50% in Group IB and 66.67% in Group IIB.


   Discussion Top


Determination of sex plays a major role of diagnosis in forensic investigations which is notoriously difficult when the body is fragmented or skeletonized as in the case of impact clashes and explosions. The tooth pulp tissue is sequestrated in a hard tissue casing where it may be protected from the detrimental effects of impact, trauma, and heat. Teeth have emerged as one of the most valuable post mortem specimens during forensic investigations because many times teeth are the only preserved remains available for forensic purposes.[9]

Because crime against children can happen anywhere, it is possible that in a forensic situation (either criminal or civil) the body of the deceased can be either left on the ground, thrown into the water bodies in a hurry, or buried in the earth, which might remain there for months or even years together.[24]

Pulp tissue from teeth is a rich source of DNA, odontoblasts, fibroblasts, and white blood cells. DNA obtained from the pulp tissue of deciduous teeth although in minute quantity can prove to be extremely helpful for sex determination with the use of adequately standardized and quantified methods of DNA analysis such as PCR technique.[12],[25]

There are several methods that can be employed for DNA analysis obtained from the pulp tissue of the teeth such as Southern-blot test, restriction fragment length polymorphism analysis, fluorescence Y chromosome test, etc.,[26] However, PCR technique has gained much popularity owing to its ability to analyze even the minute quantity of DNA, especially in cases of sex determination after months or years of death. PCR is very much cost-effective method as compared to the other methods, and in addition, it has got high specificity and sensitivity rates. Hence, amplification of DNA from a single cell, even if degraded is possible to a larger extent by PCR technique.[9]

It was also noted in the literature that the most important factors that govern proper DNA quantification and personal identification depends upon the time after death, the method of DNA extraction, and the type of soil in which the bodies were buried.[27] In the present study, we have exposed the teeth samples to two storage media, namely, natural soil and wet clay, and both these two media differ in their properties, temperature, pH, and microbial load. Our first medium of storage was the natural soil, which is composed of highly decaying organic matter and minerals. It is usually dark brown in color and is the most vulnerable to water and wind conditions. The second storage medium was wet clay. This layer is usually found in the subsurface region of soil, which consists of lots of clay, large rocks, several minerals such as aluminium, iron, and organic compounds along with water contents dripping down from the above layers.[24]

The third group of teeth samples that were analyzed immediately after extraction without exposing to any medium.

In the present study, the pulp tissue was collected from the deciduous teeth, which were in their active stages of resorption, so the quantity of the pulp tissue was less compared to that usually obtain from the permanent teeth.[25],[28] However, studies have shown that it is possible to determine the sex of an individual even if the quantity of DNA obtained from the pulp tissue was as less as 20–70 pg of good quality DNA. According to Jacqueline B. Duffy et al. (1991), owing to the active stages of resorption in deciduous teeth, the process of dehydration of the pulp varies with the apical canal diameter, which may affect the quantity of DNA obtained. It is also seen that it usually takes a time period of weeks or months for dental pulp to undergo putrefaction, degeneration, and necrosis inside an exfoliated tooth.[29] Therefore, we evaluated the usefulness of this pulp tissue stored in different environmental conditions for prolonged period of time before the PCR analysis.

The first step in the laboratory processing for the PCR analysis is the isolation of DNA, which was done using NucleoSpin tissue kit. Before moving to the PCR amplification step, it is necessary to quantify the amount of DNA present in the dental pulp tissue, so that it can be analyzed in the subsequent procedures.

On analysis of the three groups for the quantification of the DNA, the highest quantity of DNA was observed in Group III (immediately after extraction) followed by Group I (stored in soil) and Group II (stored in wet clay), which was statistically significant. This result was in accordance with the study done by Jacqueline B. Duffy et al.(1990), A. GIonesiy (1980), and S.S Raphael (1976) who stated that as the pulpal tissue dries, there is an arrest of the necrotic and/or putrefactive processes resulting in its prolonged stability and diagnostic ability.[30],[31],[32] This could be the reason why the DNA analyzed immediately after extraction at room temperature yielded a better quantity than others.

On comparison of the quantity of DNA among Group IA (stored in natural soil) and Group IIA (stored in wet clay) stored for 1 month each, the results showed a good quantity of DNA obtained from the samples stored in natural soil than wet clay, which was not statistically significant.

When the time period of storage was increased, up to 6 months in Group IB and Group IIB, the amount of DNA that could be extracted also decreased in both groups with Group II showing the maximum decrease. Among the 10 samples in Group II stored in wet clay for 6 months, the quantification could be done only for 7 samples while in Group I, 8 out of 10 samples could be quantified. This particular finding is in accordance with the study done by Suresh Vemuri et al. (2016), which also stated that the amount of DNA quantified decreases as the number of days of storage are increased.[26] Moreover, there may be other factors such as the moist conditions of soil from the wet clay, which may provide favorable temperature and humidity conditions for bacterial metabolism and growth infiltrating the pulpal space, and thus, resulting in more DNA degradation.[28]

PCR analysis of all the three groups was carried out by denaturation, annealing, and extension. On comparison of the results obtained from Group I and Group II, the PCR analysis gave many accurate results in Group I as compared to Group II, which could be attributed to the high moisture content in wet clay along with organic compounds favoring the growth of certain microorganisms enhancing the rate of decomposition of the pulpal tissue. Along with this, the pH of the natural soil ranges from 4.5 to 8.0 and that of the clay is >8.5.[30] This alkaline pH conditions of the clay enhance the decomposition rate of the pulp tissue. On prolongation of storage time, the microbial load increases that infiltrates the pulpal tissue and speeds up the degradation process and putrefies the DNA to an extent that amplification may not be possible.[36],[38]

In addition, samples of Group IB and Group IIA showed 50% false-positive results, which further decreased to 33% in samples of Group IIB stored for 6 months. These results could be attributed owing to several reasons such as the smaller quantity of pulp tissue collected and DNA isolated, contamination of pulp tissue while collection, and other human errors such as mislabeling, mixing specimens, and unspecific detection/amplification.[33] Technical issues while doing the PCR also could be one of the reasons attributed to the false-positive result obtained such as those of low annealing temperature and use of long primers.[43] On comparison of the time period of the storage in Group I and Group II, the accuracy rate of PCR analysis decreased as the time progressed.

Therefore, among the two storage media, soil proved to be a better media than wet clay owing to the fact that the DNA degradation speed, and the ability of pulp tissue in natural soil is much lesser than the wet clay even when the samples were stored for as long as 6 months.


   Conclusion Top


  • Determination of sex of teeth stored in natural soil for 1 month (Group IA) showed 100% accuracy rate that dropped down to 80% accuracy rate after 6 months of storage (Group IB).
  • Determination of sex of teeth stored in wet clay for 1 month (Group IIA) showed 88.89% accuracy rate that further dropped down to 66.67% after 6 months of storage (Group IIB).
  • Determination of sex of teeth analyzed immediately after extraction (Group III), however, showed 100% accuracy rate.
  • Samples of teeth stored in natural soil for 1 month (Group IA) had no false-positive results. However, as the time period of storage increased up to 6 months (Group IB), the false-positive rate decreased to 50%.
  • Samples of teeth stored in wet clay for 1 month (Group IIA) showed a false-positive rate of 50%, whereas the samples analyzed after 6 months of storage (Group IIB), the false-positive rate was shown to be 33.37%.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. R Manju
NITTE (Deemed to be University), AB Shetty Memorial Institute of Dental Sciences, Department of Pediatric and Preventive Dentistry, Deralakatte, Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_93_19

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