The authors inform there is no conflict of interest

Lopez Toribio Roe Mio (1)

https://orcid.org/0009-0001-3367-4920,

Castañeda Eugenio Nancy Elizabeth (2)

https://orcid.org/0000-0002-3016-663X

Manrique de Lara Suarez Digna Amabilia (2)

https://orcid.org/0000­0003­4488­252X

(1) Universidad Nacional Hermilio Valdizán, Huanuco, Peru (autor principal)

(2) Universidad Nacional Hermilio Valdizán, Huanuco, Peru (autor secundario)

E-mail: miolopeztoribio@hotmail.com

ABSTRACT

Forensic medicine is associated with the application of science to legal matters. One of the primary objectives of the judicial system is identifying a piece of evidence or an individual involved in a crime; forensic specialists play a critical role in this process. Legal odontology is one branch of forensic medicine and dentistry that focuses on using dentistry in the legal system. According to Interpol DVI regulations, dental features are one of the main components of identification. Consequently, the identification of unidentified human remains using dental features is one of the principal applications of legal odontology. The use of DNA as a human identifier, particularly in cases of severely decomposed, burned, or mutilated remains, has been made easier by recent developments in DNA technology and the use of saliva and teeth as sources of DNA. These developments also help to connect the criminal to the crime. In light of this, we provide a review of forensic genetics applications from the perspective of legal odontology here.  

In this review, results were manually extracted from articles indexed in the Scopus, PubMed, Google Scholar, and EBSCO databases that respond to the search terms that were carried out in English, Spanish, Mandarin Chinese, and French. Such as Forensic DNA, forensic genetics, legal odontology, forensic odontology, and dental DNA, to describe the medical-dental management of legal odontology to date.     

Keywords: DNA fingerprinting, legal odontology, saliva, teeth.

1. INTRODUCTION

Legal Odontology (LO) incorporates numerous study spaces, where the legal framework and dentistry overlap. This is a perceived expansion of dentistry that, in the legal field, manages the proper care and examination of dental confirmations, and the actual evaluation and documentation of dental confirmations (1). The Protection of Children from Sexual Offenses Act, 2012, the Child Labor Act, 1986, section 320 of the Indian Penal Code (IPC) for causing serious injury, and Indian Penal Code (IPC) 375 for determining the identity of the deceased and examining bite marks are just a few of the reasons forensic odontologists are essential to investigations. By offering expert testimony on dental evidence and expressing an opinion in court in compliance with the Indian Evidence Act of 1872, forensic odontologists sometimes assist the judicial system. The dentition’s distinctiveness, each tooth’s class, and unique characteristics, and the availability of ante mortem dental records all contribute to the successful identification of human remains in isolated incidents or large-scale disasters. Teeth are also thought to be a very excellent source of DNA material since they are resistant to post-mortem alterations and other environmental insults such as trauma, severe temperatures, and immersion in water (2). According to Interpol Disaster Victim Identification (DVI) guidelines, dental characteristics are considered one of the three main identifying characteristics (3).

This narrative review article aims to present the fundamental ideas of forensic DNA and highlight the domains in which DNA technologies can be used in a forensic setting, including mass disaster identification, age determination, and current suspect identification. For this purpose, results were manually extracted from articles indexed in the Scopus, PubMed, Google Scholar, and EBSCO databases that respond to the search for the terms forensic DNA, forensic genetics, legal odontology, forensic odontology, and dental DNA. Regarding the inclusion criteria, bibliographic reviews, randomized studies, guidelines, systematic reviews, and meta-analyses published between May 2013 and March 2024, in English, Spanish, Mandarin Chinese, and French were considered.

2. DEVELOPMENT OF REVIEW

DNA is the molecule that contains the biological information necessary for life, according to fundamental genetics and its use in a person’s identity. Gregor Mendel (1822–1884), an Austrian monk, stressed the inheritance of features between generations before the notion of DNA. The study of heredity and genetic differences in organisms is known as genetics. hereditary characteristics that are handed down to the next generation impact behavior, define appearance and have an impact on hereditary illnesses (4).  

Humans have 22 autosomes, or sets of chromosomes, plus one remaining pair of sex chromosomes, called XX for females and XY for males. Legal scholars should prioritize studying certain nuclear DNA (nDNA), mitochondrial DNA (mtDNA), and Y chromosomal DNA (5).

Due to the way that DNA is inherited, each parent contributes half of the autosomes, or 23 in total. From now on, autosomal analysis can be performed for individual identification, sex chromosome analysis for gender determination, and mtDNA analysis for maternal lineage (6). Like differential DNA analysis, Y chromosome determination is an expanding routine procedure in sexual abuse cases (7). This technique allows the segregation of male and female DNA and is therefore of great value in establishing the identity of the accused of a crime (8). There are around 30,000–40,000 genes known to encode different proteins throughout the human genome. “Exons” are these coding sections, while “introns” are the non-coding sections that lie between genes.  

2.1. DNA FINGERPRINTING  

Sequencing a person’s DNA is known as DNA typing or DNA profiling (5). Due to the uniqueness of the DNA pattern, sequencing is also known as DNA fingerprinting (5,8). When horrible crimes like rape, sexual assault, murder, burns, and accidents occur, DNA profiles are routinely utilized to identify the victims as well as the offenders. Forensic wildlife crimes like endangered species poaching, it is also used to settle conflicts between mothers and fathers. The technique involves collecting clean samples of the query subject and checking known sources (9). Most of the time, the samples are decomposed, adulterated, and of various unknown origins, and the discriminatory power of the DNA profiles becomes very high, with the expected probability of a match between two unconnected people being estimated at approximately 10^-10-10^-13 (5). However, issues such as DNA degradation or contamination and the closeness of victim and offender profiles (e.g., siblings) are unlikely to be recognized in the courtroom, especially when no other corroborating evidence is presented (8,9,10).

2.2. GENEALOGY MARKERS (Y CHROMOSOME, MTDNA, X CHROMOSOME)

The circular DNA helix known as mitochondrial DNA (mtDNA), which has 16,569 base pairs, is present in the cytoplasm of cells within mitochondria. MtDNA can only be inherited from the mother since mitochondria are only present in the middle and tail regions of sperm and only the head of the sperm participates in fertilization. As a result, the father’s siblings are not genetically related to him. Therefore, each sibling from the same mother has to have the same mitochondrial DNA. Thirteen proteins, twenty-two transfer RNAs, and two ribosomal RNAs are produced by the 37 genes that the nucleotides of mtDNA encode. All thirteen of these protein products are elements of the enzyme complexes involved in the oxidative phosphorylation pathway. The most typical application of mtDNA in forensics is when nuclear DNA quality and quantity are impaired. MtDNA is preferred during research due to the presence of more copies of mtDNA in a cell than nuclear DNA (5,6). Therefore, the structure and genetic constitution of mtDNA are highly conserved among mammals (6,11). However, when people are linked to one another maternally, mtDNA’s discriminating power is reduced.

X chromosome-specific genes are referred to as “sex-linked genes.” Men only have one X chromosome and will only get one copy (recessive), while women have two copies. For a woman to have a deficiency, she has to have two damaged copies of the gene (7).

2.3. GENE POLYMORPHISM

The human genome’s intergenic region includes polymorphism regions, which are repeating sequences with a high degree of variation. This may be referred to as a “length polymorphism” or a “sequence polymorphism.” These regions include DNA polymorphisms that may be used in genetic mapping and forensic investigations. DNA has two types of repeating sequences: tandem repeats and interspersed repeats, sometimes known as random repeats. Moreover, there are two different kinds of tandem repeats: short tandem repeats (STR), often called “microsatellites,” and variable number tandem repeats (VNTR), also called “minisatellites” (1,5,10). The primary repeat units of STRs are 2 to 6 base pairs, whereas those of VNTRs vary from 10 to 100 base pairs (bp). Both STRs and VNTRs are useful tools for determining DNA, but since STRs are more abundant in the genome than VNTRs, characterizing them is easier. The foundation for genetically identifying a person and defining its genetic profile is the variation in loci, which are nothing more than repetitive sequences, between people. Length polymorphism is the term for when there are differences in the number of tandem repeat units. Point mutations, sometimes referred to as single nucleotide polymorphisms (SNPs), are variations in a single base pair of a sequence (1,5,10).

2.4. BIOTECHNOLOGIES FOR FORENSIC DNA ANALYSIS

Additionally, new technologies including massively parallel sequencing, and microfluidics, are often heralded as one of the most important scientific advances in molecular biology, such as integrated fast polymerase chain reaction (PCR) systems, and real-time PCR represent significant advancements in PCR technology, enhancing the speed, accuracy, and convenience of DNA amplification and analysis in various applications, from clinical diagnostics to research and forensic investigations. To help with data processing from these sophisticated analytical tools, expert systems have also been created (12). Technological developments in forensic DNA detection were greatly aided by Kary Mullis’s discovery of the PCR in 1986. DNA fingerprinting was created by Alec Jeffrey in 1986, and forensic investigators were the first to use the method. The first step in processing evidence in a laboratory setting is to extract the DNA molecule from the biological material (13).PCR-based analysis has the advantage of being highly sensitive and less time-consuming (14).

2.5. FORENSIC DNA FINGERPRINTING

Among the initial forms of forensic DNA profiling, the application of restriction fragment length polymorphism (RFLP) was the first (12,15). This method involves cleaving certain locations along the DNA sequence using the enzyme restriction endonucleases. Following agarose gel electrophoresis to separate the resultant DNA fragments by size, the fragments are transferred to a nitrocellulose slide, hybridized with locus-specific probes (chemiluminescent or radioactive), and then detected as bands by autoradiography or chemiluminescence. RFLP requires a large amount of DNA and is rarely used in forensics today. The invention of PCR is also one of the other factors that have led to its degradation in forensic medicine (12,15).

3. AUTOSOMAL SHORT TANDEM REPEAT (STR) PROFILE

Five to ten percent of the human genome is made up of sequences that repeat themselves. A section of DNA known as the STR is home to many tandem repeats. The DNA sequence in such STR units is 2 to 6 base pairs, and there are more than 105 STRs in the human genome (1,16). The number of tandem repeat units at the STR locus differs from individual to individual and therefore determines the genotype for human identification (Fig. 1). A minimal quantity of DNA (about 1 ng) is required for the profiling of STRs, which is present on all chromosomes, including sex chromosomes. This amount is 50 times less than that needed for RFLP analysis. This 1 ng of DNA sample may be amplified using the PCR technique to complete a STR profile. Additionally, PCR amplification of multiple STR loci can be performed simultaneously in the same PCR tube (1). This method is called multiplex PCR or multiplexing. STR markers that are sold commercially are often used in forensic DNA profiling. For instance, CODIS (Combined DNA Index System) is the name given to the top 13 STR markers created by the FBI in the United States (1). According to Malik et al. (1), the STR locus is of three types (fig. 2):

1. Simple STR: the repeating units are of identical length and sequence.
2. Composite STR: consists of two or more adjacent simple repeats.
3. Complex STR: has several repeating blocks of different unit lengths and variable intermediate sequences.

Fluorescent dye-labeled primers are employed to amplify the STR loci after DNA extraction from the sample. The amplified products are sorted and located using capillary electrophoresis after a few PCR cycles. The data is generated by the computer and shown as peaks on an electropherogram. Each DNA fragment-specific dye is found by the detector. A peak represents each DNA fragment that has been found. The dimensions of each peak are determined using allelic ladders and size criteria. Every allelic ladder is appropriately solved to determine the correct STR allele (1).

4. SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS)

Variations that occur between individuals in a single base sequence at a particular point in the genome are called a single nucleotide polymorphism or SNP (16). SNPs are the most common form of genetic variation in humans. It can be the result of a substitution, deletion, or insertion of bases at a single site. They occur every 100 to 300 bases along the DNA strand. Across the human genome, around 1.4 million SNPs have been identified and therefore have the potential to be used as markers for forensic applications (Fig. 3). The advantage of SNPs in forensic applications over STR profiles is that PCR amplification of the SNP marker can better resist the degraded DNA sample and can be multiplied to a higher level than STRS (1,17). To achieve enough discriminating power, however, a substantial number of SNPs must be analyzed. While SNPs are not likely to take the role of STR analysis, they may be a useful supplementary tool in difficult forensic instances (18).

5. “THE ORAL CAVITY IS THE BLACK BOX OF THE ORGANISM” IN FORENSIC INVESTIGATION   

Interdisciplinary expertise is required in legal dentistry because information from the oral cavity may be used to identify a person or offer details required for legal proceedings. Furthermore, data obtained from the oral cavity can limit the search range of an individual and play a key role in the victim identification process after disasters or mass catastrophes (1,19). The oral cavity is a rich and non-invasive source of DNA and can be used for the identification of people and to provide information necessary in legal proceedings (1,19).

5.1. TEETH ARE THE MAIN SOURCES OF DNA FOR FORENSIC RESEARCH   

The bodily fluids submitted for DNA analysis in forensic investigations are often semen, blood, and saliva. However, teeth are the tissue that offers the best chance of extracting DNA from highly decayed, skeletonized, or burned individuals. In medicolegal death investigations, the source of DNA can be divided into two areas: first, DNA obtained from the outside of the body or from inside the body cavities (e.g., blood, saliva, semen, fluid vaginal, etc.), secondly, DNA from biological materials (for example, liquid blood, bones, teeth, nails, etc.) (19). Genetic information is abundant in the nucleated cells of teeth and the periodontal ligaments that surround them. The tooth is crushed or sectioned at different levels to extract DNA after a thorough morphological and radiological examination for identification and age estimate, respectively (20). One of the most recommended methods for tooth DNA extraction is cryogenic polishing by sectioning the tooth at the cement-enamel junction for a conservative approach to dental DNA (21). Figures 4 and 5 represent the sequence of DNA extraction from the teeth. Pulverizing or crushing the entire tooth into a fine powder can produce enough DNA for research (1). However, during such investigations using teeth, recording other tooth data, for example when exposed to intense heat, the internal structures of teeth, such as the pulp cavity, may protect genetic material. The enamel and dentin layers can insulate the inner parts of the tooth, allowing for the extraction of DNA from dental pulp for identification purposes. Likewise, the research describes an alternative method to recover DNA from molar teeth (22,32[DC4] ).  

5.2. DNA FROM SALIVA AND BUCCAL MUCOSA FOR FORENSIC RESEARCH

The most easily accessible objects for saliva recovery may include nonliving items including apparel, food items, cigarettes, cigars, other smoking accessories, dental equipment, beverage containers, postal stamps, and envelopes. When it comes to processing a bite mark for DNA, the specific methods of the labs that process the genetic material are often followed for the extraction of salivary DNA from personal effects. For DNA analysis, exfoliated oral mucosal epithelial cells are collected using the buccal swab procedure. Authorities conducting investigations may establish a connection between the offender and the victim by using saliva samples taken from the crime site and analyzing the samples using DNA. For DNA collection, the buccal swab is a rigorous and cautious substitute for blood (23,34).

5.3. SALIVARY DNA IN BITE MARK EVIDENCE FOR FORENSIC RESEARCH

The use of different light sources may increase the visibility of saliva stains in situations of sexual assault when bite marks are used as evidence. This preserves the DNA sample unaltered and enables analysis to be done. assessment even in situations in which there isn’t a confirmed injury. While white blood cells, mostly from the gingival crevicular fluid, and cells lost from the oral mucosa do carry DNA, saliva does not (24,25).

In 1992, a method to obtain saliva for DNA analysis was well documented, but in 1997, Sweet et al. used the double swab for the collection of salivary DNA from the skin, which eventually became a successful method for the collection of salivary DNA (26,27,28). In practice, to collect material using oral mucosa, a double swab technique is followed in which the mouth is rinsed with water and then the oral mucosa is scraped with sterile gauze. and allowed to dry and then scraped the mucosa twice with two different swabs followed by allowing the two swabs to dry in the open air, after which they are placed in a labeled envelope and sent to the laboratory (1). The washing action of the rinsing [DC5] leads to the elimination of possible contaminants and improves the quality of the DNA thus obtained (29).

The application of dental parameters in the identification of people is significant, but in practical situations, it is not fully proven. This is probably because there is a dearth of knowledge among those involved in the legal and police systems, there isn’t a formal team for identifying victims of disasters that includes a certified forensic odontologist, and records are either poorly maintained or nonexistent. dental discoveries obtained before death in the nation’s clinical dentistry area (30,31,32). To verify the identification of an unidentified corpse, police, and legal authorities therefore only need secondary characteristics such as personal possessions, tattoos, etc. in addition to DNA testing. In some situations, teeth serve as the sole source of DNA, and in such situations, ordinary dentists or forensic odontologists do not remove the “ideal tooth” for DNA. DNA evidence may be gathered from remnants of the perpetrator’s saliva on the indentation (bite mark) on the victim’s body in cases of sexual assault including bite marks. DNA analysis is essential in connecting the culprit to the crime scene (31,33,34). At this point, it is vital to note that acquiring DNA evidence from the assault bite mark (if any remains on the victim’s body) and collecting a saliva sample from it immediately for examination are both crucial. Consequently, the tooth and saliva are valuable materials for forensic DNA studies from the standpoint of legal dentistry (31,34).

6. CONCLUSIONS

There has been interest in using DNA techniques in forensic investigations due to the growing accessibility of DNA technology. A revolution in forensic medicine, including legal dentistry, has resulted from this. As the two main techniques of identification in IVC, dental analysis is positioned in between fingerprint and DNA analysis. From the standpoint of legal dentistry, anyone working in the field must possess some theoretical and practical understanding of DNA analysis. Saliva in bite marks is sometimes disregarded as a potential source of DNA in instances of sexual assault. Since it changes according to the circumstance, players in forensic medicine are likewise unaware of the best tooth for DNA extraction in most situations. Potential techniques for detecting the specific tooth’s age or sex may be tried and documented before the tooth is destroyed to retrieve DNA. This study aims to provide key information on DNA and DNA analysis, particularly for tooth and saliva sources, to those involved in the legal and forensic systems.    

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