Forensic Dentistry – the key to the truth

Summary Introduction Human organism can be identified through testing and analysis of DNA sequences. The most common source of DNA for analysis is blood, soft tissues, hair, bones and teeth. Teeth represent a tissue of choice for analysis in those cases where there is high degree of degradation of other tissues. Hard tooth structure provides protection and preservation of DNA molecules. The aim was to investigate which group of teeth and dental tissue (pulp or hard dental tissues) has the greatest amount of DNA. Material and method Forty-five extracted teeth were analyzed. In the first examination 30 teeth were divided into the three groups (10 teeth each): first group were incisors, second premolars and third molars. The teeth were measured before and after the procedure of DNA isolation using special scale with precision of 0.02-0.000005ng. The procedure included grinding teeth in a blender and DNA isolation using commercial kits (isolation with magnetic particles). For the second test 15 teeth divided into two groups were used. In the first group isolation of DNA molecules was performed from pulp tissue, and in the second group from hard dental tissues. The quantification of samples was done with Quantifiler® Duo DNA Quantification Kit by Applied Biosystems. Results The greatest amount of DNA was obtained from molars (0.230011ng/μl/g) while the smallest amount of DNA was obtained from incisors and it was 0.06437ng/μl/g. In addition, the amount of DNA isolated from pulp tissue was significantly greater than that from hard dental tissues (pulp of molars obtained quantitatively the largest amount of DNA). Conclusion Main tissue to be used for the isolation of DNA from a tooth is pulp, but in those cases when it is not present (endodontic treatment), hard dental tissues provide sufficient quantity of DNA for identification procedures. The group of teeth that provides the greatest amount of DNA is molars.


INTRODUCTION
Forensic dentistry represents a scientific discipline in which field of interest is the tooth and all its features, starting with its morphology and analysis of its structural components, in order to get information to solve criminal cases or use in court (legal) system. Forensic odontology or Forensic dentistry is defined by Keiser-Neilson in 1970 [1] as "the branch of forensic medicine that in the interest of legal system handles and examines dental evidence and appropriately evaluates and presents dental findings". The most common role of forensic dentist is identification of deceased individuals [2]. Dental identification includes two modes. The first one is comparative identification used to verify that the remains of a deceased person and the person represented by dental data obtained before death (antemortem) are one and the same individual. The second method includes cases where data before death (antemortem) does not exist. In that case, forensic dentist complete data after death (postmortem) obtaining the deceased person's individual characteristics in order to compare them with antemortem data. This process is called postmortem dental profiling.
Human body can also be identified through testing and analysis of DNA sequences. Each cell of an individual contains multiple DNA copies. Every human being is characterized by a unique (single) DNA sequence due to hypervariable regions of DNA that are specific for that person. The sequence of DNA molecules called base pairs (bp) in each individual varies with the exception of identical twins and is called sequence polymorphism. Length polymorphism as AATG-AATG (repeat twice), or AATG-AATG-AATG (triple repetition), called Short Tandem Repeats (STR) is used in forensic identification [3,4]. These repetitive sequences are called DNA fingerprints and the procedure for their identification is called DNA typifying, i.e. profiling (DNA Profiling). DNA profiling represents standard procedure in forensic system for human identification, criminal cases and determining fatherhood [5].
Beside DNA isolation from hair, bones, dried soft tissue, teeth give best usable DNA for identification because of the hard dental tissue that physically protects the pulp. Most often tissue used for the isolation of DNA is pulp tissue, which is connective tissue composed of cells (odontoblasts, fibroblasts, endothelial cells, peripheral nerves, undefined mesenchymal cells, macrophages and lymphocytes). However, in cases of endodontically treated tooth, where pulp is removed, DNA may be extracted from calcified tissue, dentin or dentin and cement together by the milling procedure to produce powder. DNA isolation is also possible from periodontal fibers and attached periodontal bone fragments.
The quality and quantity of extracted DNA from teeth depends on several external and individual factors. From individual factors, special attention is given to the volume of pulp chamber that is different in different teeth groups. Like for other tissues, DNA isolation from teeth depends on external influences like temperature, time of death, humidity, bacterial and fungal contamination [6,7]. Also, the fastest DNA molecule degradation occurs in the first two years, depending on external and personal factors [6].
The aim was to investigate which group of teeth and dental tissue (pulp or hard dental tissues) has the greatest amount of DNA.

MATERIAL AND METHODS
Forty-five extracted teeth without major caries damage (in vitro study) extracted at the Clinic for Oral Surgery at the University Dental Clinical Center-St.Pantelejmon in Skopje were analyzed. DNA analyses were conducted at the Institute of forensic medicine, Criminology and Medical deontology. The method with magnetic particles as solid phase for binding the DNA molecule was used (Prepfiler Kit from ABI ™).
This technique of DNA isolation (unlike the old process of organic isolation) is based on solid phase extraction (SPE). It uses columns filled with silica (or other solid phase) that selectively bind (adsorb) nucleic acids (DNA or RNA) depending on the environmental pH and concentration of salt in the buffer (in our case the solid phase were magnetic particles). The columns and all buffers used to perform the procedure come in the form of commercial kits and the procedure was performed according to the manufacturer's instructions.
Quantification of samples was performed using the Quantifiler DNA Quantification Kit (Applied Biosystems) on 7500 Real Time PCR System. This approach allows DNA quantification in the initial sample by measuring the fluorescence signals during amplification of the targeted sequence. The primary objective of the Real Time PCR quantification is determining "amplifiable" DNA present in the sample. The input amount of Quantifiler® Duo Primer Mix for quantification of one sample was 10.5 µl, and the input amount of Quantifiler® Duo PCR Reaction Mix for quantification of one sample was 12.5 µl. The volume of a sample added into the reaction for quantification was 2 µl. The full volume of a reaction mix was 25 µl.
The first examination comprised 30 teeth divided into the three groups of 10 teeth: incisors, premolars and molars. The procedure of DNA isolation from teeth included teeth grinding in a blender and use of commercialized kits (isolation with magnetic particles). With this method, teeth that had previously done endodontic treatment could not be used for DNA isolation [8]. First, the teeth were rinsed under the tap water and surface was well cleaned with a scalpel and a brush. In addition, the teeth were kept in ultrasonic bath in 1% sodium hypochlorite (NaOCl) twice for 10 minutes with replacement of NaOCl solution. This procedure was repeated also twice with distilled water. The cleaning procedure took about 40 minutes. The teeth were placed then in a fume cupboard to dry overnight. Dried teeth were weight first and then ground in a blender for small bones and teeth to obtain tooth powder.
For the second test 15 teeth divided into two groups were used. In the first group isolation of DNA molecules was performed from the pulp tissue, and in the second group the same teeth that pulp was collected from were grinded and used for DNA isolation. Pulp was collected using nerve extirpators after performed horizontal sectioning of the tooth. This procedure of horizontal separation of the tooth in the line of the neck of the tooth was done in order to preserve occlusal surface of the tooth that can serve for further procedure of identification. This separation was done using diamond burs and micro motor. The bur was moved around the teeth in circular motions. When close to pulp chamber, teeth were split with lever, to avoid overheating and mechanical damage of pulp tissue, and thus contamination of the material.

RESULTS
In our first test the analysis and comparison of different groups of teeth, freshly extracted incisors, premolars and molars was done. Those are three groups differing in size and number of roots, i.e. there are large morphological differences between them. The results are shown in Tables 1-3.
In the second test the amount of DNA obtained from the pulp and hard dental tissues was compared. The results are shown in the Tables 4-5.

DISCUSSION
In forensic cases of identification, where human remains are extremely damaged or degraded, teeth and bones are often the only source of DNA [9, 10]. Genetic analysis of teeth depends on the amount of DNA present, the level of degradation and efficiency of the collecting processes of DNA and methods used for DNA isolation [11]. With the current knowledge about the structure of teeth and after death degradation of DNA in dental tissue, our paper offers optimal selection of teeth, in order to increase the effectiveness of DNA extraction and DNA profiling.
It can be seen that the greatest amount of DNA was obtained is in the group three or group of molars and it was 0.230011 ng/μl/g tooth, while the smallest amount of isolated DNA was obtained in the first group, i.e. the incisors and it was 0.06437 ng/μl/g tooth. The concentration of isolated DNA from the group of molars was four times greater than group of premolar and five times than group of incisors. Comparison of the first and second group, i.e. group of premolars and incisors indicated approximately the same concentration of isolated DNA, with slightly more from the group of premolars. This can be explained by the fact that premolars have variations in the crown size and number of roots (one or two roots). Positive correlation was found between the tooth weight and the amount of isolated DNA. The lowest weight had incisors with average of 0.605g, and they gave the least amount of isolated DNA. Then, premolars had average of 0.892g, and they provided more isolated DNA, while molars weighing 1.705 g gave the greatest amount of isolated DNA. Other studies that compared the amount of DNA between different types of teeth showed that teeth with the largest quantity of dental tissue are the best source of DNA [11,7]. It has also been confirmed that greater amount of DNA can be obtained from teeth with more roots and this is due to the larger root surface that provides greater amount of cement [11].
Teeth with more roots not only have larger root area and higher amount of pulp tissue in which odontoblasts and cementocites are located, but also show more cellular cement, compared to other groups of teeth, which in some cases have little or no cellular cement. Therefore, molars would be teeth of choice of tooth for DNA sampling due to the largest surface area of the root. In the absence of molars, premolars are recommended for DNA isolation, because they have greater amount of cellular cement than anterior teeth, but canine would have larger volume of pulp. Greater amount of cellular cement provides greater number of cementocites, cells that are the main source of DNA.
In the second part of the study, it was shown that the amount of DNA obtained from the pulp was significantly higher than the amount of DNA obtained from hard dental tissues. The amount of DNA obtained from the pulp, by different groups of teeth, indicated that the group of  molars quantitatively provides the largest amount of DNA. This is due to the fact that pulp tissue of molars has the largest number of present cells that are major source of DNA (Table 5). The quantity of DNA obtained from pulp complex of three different groups of teeth, showed that molars provided the largest amount of DNA. That was expected, as the volume of pulp chamber in molars is the largest. Group of premolars and incisors provided similar amount of isolated DNA that was six times smaller than from the group of molars. Similarly, the amount of DNA isolated from hard dental tissues was significantly higher in the group of molars than the other two groups (premolars and incisors) that were similar. Comparing the amount of isolated DNA (ng/μl/g) from pulp and hard dental tissue showed that pulp provided 85 times higher amount of DNA.
Dentin / pulp complex constitutes the bulk of the tooth, and unlike the enamel, is highly complex cellular tissue. The pulp is rich vascularized and innervated, and contains connective tissue with various types of cells. These include odontoblasts (cells that produce dentin), fibroblasts, defense cells (macrophages and histocytes), plasma cells, nerve cells, undifferentiated mesenchymal cells [12]. Knowing that about 80 diploid cells are sufficient to provide a minimum amount of DNA required for STR mapping, it can be concluded that the pulp is an extremely valuable source of DNA. Pulp complex is in connection with periodontal tissues (tissues that connect any tooth to the alveolar bone) over the top of the root, and through accessory canals [13].

CONCLUSION
Pulp tissue and cement are clearly the most valuable sources of nuclear DNA from the tooth. Enamel protects dentin and pulp, but has no DNA. Main tissue that should be used for DNA isolation is pulp, but in those cases where it is not present (endodontic treatment), hard dental tissues provide sufficient quantity of DNA for identification procedures. The group of teeth that provides the greatest amount of DNA is molars.