Human Identification

The problem of identifying individuals for the purpose of establishing biological relationships between two or more individuals has long been a topic within families and has resulted in many inconsistencies. Using DNA samples from individuals, identification tests are the most accurate method for determining the biological father in the field of Abbott or forensic medicine, a process that is facilitated by a blood sample or any other specimen of human biological origin. When the paternal rights and responsibilities come into play and doubts regarding the father-child relationship arise, the Paternity test can acquire particular significance. Multiple motives underlie the undertaking of such genetic tests including the acquisition of legal rights pertaining to child custody, the identification of deceased individuals and war martyrs, the establishment of inheritance claims as well as the determination of familial relationships.

Paternity or kinship tests encompass the analysis of the DNA of each person to obtain genetic profiles or genetic fingerprints. The comparison of the information derived from two individuals enables the determination of the relationship between them. In the past, alternative methods such as blood group determination and human leukocyte antigen (HLA) examination were employed to investigate kinship relationships, but these approaches have now been supplanted by the employment of DNA profiling, which provides notably enhanced accuracy.

On the whole, each individual inherits half of their genetic material from their father and the other half from their mother. Within the entirety of the human genome, only approximately 2% of genes encode for proteins. Within an individual’s genome, there exist repetitive sequences that are distinct and it is possible to ascertain the kinship relationship and identity of individuals through the assessment of these markers. DNA profiling or genetic fingerprinting is the process of identifying individuals based on their genetic characteristics. DNA profiling stands as a practical technique within the realms of forensic medicine and criminal investigation, as it allows for the comparison of genetic markers of suspected individuals with those of another person (e.g., the victim) in order to ascertain the identity of the perpetrator or criminal.

Among the markers that are widely employed in discussions of identity determination, microsatellite markers known as Short Tandem Repeats (STRs) hold a prominent position. These markers consist of repetitive sequences of 2 to 7 nucleotides (often 4 nucleotides), and the number of repeats (n) within the STR marker varies and proves unique to different individuals, thereby rendering them suitable indicators in the determination of individuals’ identities. Additional crucial markers in identity determination include the SE-33 marker, mitochondrial DNA, and the Amelogenin gender determination marker, all of which are simultaneously amplified within a multiplex polymerase chain reaction (PCR).

Samples for Human Identification

Blood
Nails
Amniotic fluid to determine the identity of the fetus
Hair
Corpses bones
Saliva
CVS sample
Tooth

Types of markers used in human identification

For human identification various markers are used, which will be introduced later.

STR markers

STR markers, short tandem repeats (STR), are brief DNA sequences consisting of approximately 2 to 6 nucleotides. These markers constitute roughly 3% of the human genome. The number of repeating units within individuals exhibits significant variability, thereby enhancing the efficacy and precision of identification and recognition. It is currently understood that STR markers are non-coding in nature and certain studies have indicated that these repetitive sequences may possess diverse mechanisms in governing gene expression and the manifestation of distinct phenotypes. The Federal Bureau of Investigation (FBI) in the United States has designated 13 original CODIS STR markers which are now commonly employed for individual identification within the nation. CODIS, or the Combined DNA Index System, constitutes the national DNA database in the United States encompassing three tiers of information, namely:

The first level: Local DNA Index Systems (LDIS) serves as the repository of DNA profiles.
The second level: State DNA Index Systems (SDIS), facilitating the exchange of information among domestic laboratories in the United States.
The third level: National DNA Index System (NDIS), enabling different states within the United States to compare their DNA data with one another.

The CODIS software encompasses a myriad of databases, which vary depending on the nature of the desired information. These databases encompass diverse categories such as individuals who have gone missing, convicted criminals and forensic samples collected from multiple crime scenes. Previously, up until the year 2017, CODIS incorporated a set of 13 autosomal loci STR markers, whereas this number has now been augmented to 20 markers.

These CODIS markers, which are situated within a gene, are denoted by the name of the corresponding gene. To illustrate, the TPOX marker is situated within the human thyroid peroxidase gene. Conversely, for markers that are not linked to a specific gene, a distinctive nomenclature system has been devised. Under this system, a marker on the second chromosome is denoted as D2, with “D” representing the chromosome symbol and the subsequent number indicating the chromosome number. Following the chromosome number, the letter “S” is appended, with the ensuing numbers indicating the position of the marker. The 20 primary CODIS STR markers are as follows:

CODIS Markers
D8S1179	D7S820	D5S818	D3S1358	CSF1PO
FGA	D21S11	D18S51	D16S539	D13S317
D2S1338	D2S441	TH01	TPOX	vWA
D22S1045	D1S1656	D12S391	D19S433	D10S1248

Y-STR marker

Y-STR markers are repetitive sequences known as short tandem repeats (STRs) that are situated on the Y chromosome within the male. Due to its exclusive presence in males, Y-STR is frequently employed in establishing paternity and male identification, albeit with a narrower scope of analysis compared to markers found on autosomal chromosomes. These markers serve to illustrate the heterogeneity among male individuals within society. The majority of Multiplex Y-STR kits investigate various loci on the Y chromosome. This particular class of markers follows a nomenclature convention commencing with the acronym DYS (DNA Y-chromosome Segment), with subsequent numbers denoting the locus’s position, such as DYS455, whose sequence is AAAT.

Mitochondrial DNA marker

Mitochondria, one of the cytoplasmic organelles present in eukaryotic organisms, plays a vital role in cellular energy production. Its circular DNA comprises 37 genes, and cells may possess one or more of these organelles. The mitochondrial genome also encompasses a region known as the control region which contains two highly variable sequences, namely HSV1 (342 nt) and HSV2 (286 nt). The rate of alteration in the HSV1 and HSV2 regions is remarkably high, making them effective tools for individual identification. Moreover, the mitochondrial genome exhibits remarkable stability against heat due to its high GC content.

Mitochondrial DNA (mtDNA) has useful markers in the field of forensic medicine and identification of people due to its high copy number, maternal inheritance and low recombination rate. While the identification of individuals for forensic purposes typically relies on genetic profiling using nuclear genomes and STR markers, there are instances where autosomal STR markers may be severely degraded or the DNA quantity extracted from a sample is insufficient. In such cases, mitochondrial markers can serve as a reliable means of establishing identity.

Amelogenin Marker

Genotyping of the amelogenin gene which located on the X and Y chromosomes, is currently employed for the purpose of DNA profiling. This is carried out in order to ascertain and establish the identity of individuals. The amelogenin gene, found solely on the sex chromosomes X and Y, possesses a single copy on the X chromosome (AMELX) at position Xp22.1-Xp22.3 and another copy on the Y chromosome (AMELY) at position Yp11.2, both of which exhibit a high degree of sequence conservation. Considering that this gene is located only on sex chromosomes X and Y, it can be used to determine gender.

Primers are designed to bind to the first intron in the amylogenin gene on both the X and Y chromosomes, thereby facilitating the amplification of two regions that possess identical sequence lengths. In comparison to AMELY, AMELX exhibits a 6 bp deletion within intron 1. Consequently, the amplified fragment originating from the X chromosome consists of 106 base pairs, while the corresponding fragment arising from the Y chromosome comprises 112 base pairs. The presence of both these products indicates the male genotype, whereas the presence of a single product indicates the female genotype.

SE-33 marker

The SE-33 marker, which is frequently examined in STR kits for Human identification, is one of the markers. The SE-33 marker also known as the ACTBP2 gene, is situated at the 6q14 position on chromosome 6. This particular locus, SE-33, is characterized by its high level of polymorphism with a total of 53 discernible alleles and 292 genotypes observed among a population of 938 unrelated individuals in the United States. Given the genetic diversity and polymorphism exhibited by this gene, it is now widely recognized as a significant marker in the process of ascertaining the identity of individuals through DNA profiling.

Types of prenatal identification tests

In numerous familial matters, there arises a necessity to execute the Paternity test between father and fetus, which may be accomplished through a range of techniques to procure a fetal sample. Some prenatal identification methods are invasive while others do not. These techniques encompass the following:

Amniocentesis (invasive technique): in this method, the required DNA sample is obtained by collecting some of the amniotic fluid around the fetus. This method presents a risk of abortion and is bound by a temporal constraint (from the 15th to the 20th week of gestation).
NIPT test (non-invasive): This test employs the maternal blood sample that encompasses fetal DNA for the purpose of conducting the paternity test.
CVS method (invasive): This method entails the collection of placental tissue, typically performed within the 10th and 13th weeks of pregnancy and there is a risk of miscarriage.

Techniques used in human identification

Various methods can be used in the field of paternity tests, such as multiplex fluorescent PCR and RFLP.

Multiplex fluorescent PCR

Multiplex PCR is executed through the utilization of primers that are labeled with fluorescent dyes. This approach proves to be uncomplicated, precise and sensitive for the purpose of conducting paternity tests. In Trita Human Identifier Kit®, the method of multiplex fluorescent PCR is employed alongside the usage of internal allelic ladder to amplify gene markers. Within these kits a pair of primers is utilized for each marker, of which only one is labeled with a fluorescent color. The internal allelic ladder is regarded as a qualitative standard indicator in establishing the accuracy of genotyping STR markers and it is integrated as one of the constituents of the kit solution.

In general, allelic ladder is a mixture of highly specific alleles in terms of sequence and size. This ladder is created utilizing primers that are similar to those used for the tested samples and it is employed during capillary electrophoresis to precisely identify alleles. In view of the fact that different equipment and conditions are employed in laboratories, it is advisable to employ the allelic ladder when assessing STR markers. Given that capillary electrophoresis or capillary electrophoresis technique separates replicated DNA fragments based on the difference of one nucleotide, the use of the allelic ladder in each run of capillary electrophoresis is imperative.

RFLP

The RFLP (Restriction fragment length polymorphism) technique is utilized for the examination and analysis of polymorphisms specifically those involving a single nucleotide (SNP). This method relies on the utilization of enzymes with limited activity to fragment the sample, followed by the separation of these products using gel electrophoresis. In the context of paternity disputes, the RFLP method serves as a suitable technique for exploring genetic differences between individuals.

MLPA

MLPA (Multiplex ligation-dependent probe amplification) is another PCR-based method that is used to detect STR markers as well as polymorphisms. This technique is performed in the form of Multiplex PCR and allows the user to examine multiple regions of the individual’s genome with up to 40 different probes. MLPA probes are two half-probes, 3′ half-probe and 5′ half-probe, one of which is complementary to a specific target sequence and the other is a universal primer, which enables the simultaneous amplification of multiplex PCR for all probes.

In addition, one or both halves of the probe contain a stuffer sequence, which allows the separation of the fragments using electrophoresis from the length of the probe itself and thus the different size of the amplified product. The half-probes used will lead to the amplification of that part of the DNA only if they match perfectly with the target sequence.

Considering that only the probes whose ligation process has been completed can be amplified during PCR, the number of products resulting from the connection of the probes is a measure of the number of target sequences in the sample. After amplification, PCR products will be separated by size using capillary electrophoresis under denaturing conditions. By measuring the height or area of fluorescence peaks derived from PCR, the amount of PCR product after normalization and comparing it with control DNA samples, as a result, the relative amount of the target DNA sequence in the input DNA sample is determined.