Discovery and development of forensic genetics took a long time and required a lot of field practice. After the discovery of the ABO blood types, scientists start to use blood groups in identification for forensic genetics. In 1910, the French criminologist Edmond Locard proposed the Locard’s exchange principle and stated that “every contact leaves a trace,” which laid the foundation for modern forensic science (1). In 1953, the discovery of the double-helical structure of DNA enabled the start of forensic genetics research at the molecular level (1). Deoxyribonucleic acid is the basic and fundamental element in almost all life forms. In 1984, Sir Alec Jeffreys, a British geneticist studying inherited diseases, discovered DNA’s ability to individualize humans (2). The use of DNA in forensic sciences relies on the fact that every person has its own unique DNA, except for monozygotic twins. Testing techniques are mostly built on parts of DNA that can make differences among different individuals. The locations that make differences among individuals are called polymorphisms and referred as markers in forensic genetics. Forensic genetics uses genetic markers for identification. The development of genetic markers has gone through four major stages characterized by the use of morphological markers, cytological markers, biochemical markers and molecular markers (1).
Restriction Fragment Length Polymorphism (RFLP)
Testing techniques were developed over the years with new findings in the area of forensic genetics. One of the first techniques used for identification was Restriction Fragment Length Polymorphism (RFLP). Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases (3). Technique involves digestion of the DNA sample with restriction endonuclease enzymes. After the digestion, fragments are separated according to their sizes using gel electrophoresis. After gel electrophoresis, denaturation of the DNA takes place. Southern Blotting process can be used after denaturation as explained in Figure 1.
The number of repeats affect the length of the resulting DNA. Scientists use this technique by comparing the lengths of the strands for identification. This technique was replaced with new ones because the process was extremely time consuming.
Variable Number Tandem Repeat (VNTR) Analysis
The other technique used for identification is Variable Number Tandem Repeat (VNTR) analysis method. A variable number tandem repeat (or VNTR) is a location where a short nucleotide sequence is organized as a tandem repeat and is called a minisatellite. This tandem repeats mostly show difference in length among people. Basic explanation of this technique is to amplify the DNA with Polymerase Chain Reaction (PCR), digest it with restriction enzymes that cut around repeated parts of DNA. After the digestion process, gel electrophoresis technique is used to see the bands from all of the DNA. Southern Blotting method is used for the selection of the bands that represent the variable number tandem repeat alleles. Since this number of repeats in each person is different, it can be used for identification. Figure 2 represents an example of Southern Blot DNA fingerprint. In Figure 2, Locus A is a tandem repeat of the motif GC and the four alleles with two (A2), three (A3), four (A4), five (A5) repeats are shown (5). Locus B is a tandem repeat of the motif AGCT and the two alleles with two (B2) and three (B3) repeats are shown (5). Individual #1 is heterozygous at Locus A (A2 / A5) and homozygous at Locus 2 (B2 / B2: note that this genotype gives a single-banded phenotype in the fingerprint) (5). Individual #2 is heterozygous at both loci: (A4 / A3 and B3 / B2) respectively). The two individuals are distinguishable at either locus (5).
Short Tandem Repeat (STR) Analysis
Nowadays, most frequently used and worldwide technique for identification in forensics rely on short tandem repeats (STRs). A Short tandem repeat is called a microsatellite which has 2 – 7 base pair length repeats. Short tandem repeat numbers are also varying between each individual making this technique useful for identification. The difference between the Restriction Fragment Length Polymorphism (RFLP) and Short Tandem Repeat (STRs) is that the restriction enzymes are used in RFLP. PCR is used in STR. Variability of STR regions allows making a discrimination between one DNA profile and another. In STR analysis, sequence specific primers are targeting STR loci and amplification is done by PCR. For separation and detection of DNA fragments, gel electrophoresis or capillary electrophoresis (CE) can be used. Y-STRs are short tandem repeats that are found on the Y chromosome. Y-STRs are used for sex identification in crime scenes which have mixed male-female samples, paternal identification and family searches.
Main advantage of STR analysis is their high allele diversity. The most polymorphic STRs have a high discriminating power (probability that two randomly selected individuals have distinct genotypes) and low probability of match (probability that two randomly selected individuals have identical genotypes) (7).
The main disadvantage of this analysis is that STR analysis is comparable meaning that there has to be reference samples to compare. If there is not a reference sample to be compared, the other option is to search DNA databases of suspects. In Unites States national DNA database is called Combined DNA Index System (CODIS). CODIS has DNA profiles obtained from crime scene evidences.
Another disadvantage comes in such situations when evidences from crime scenes are very degraded and it is very difficult to obtain a reliable data for evaluation. In face of such difficulties using traditional STR markers protocols in some situations, various researches have allowed the use of genetic predictions of externally visible characteristics (EVCs) to be of assistance in police investigations, in both tracking suspects and identifying victims (7).
Certain variations and single nucleotide polymorphisms (SNPs) in DNA can cause amino acid substitutions and these substitutions can alter the functional properties of proteins that are translated. Functionally altered proteins can consequently be expressed as distinct phenotypes that can make the visible characteristics of a person. A lot of studies have been conducted by scientists in order to obtain information about an individual’s physical features from DNA extracted from a blood drop or hair strands. In these studies, it is concluded that there are certain polymorphisms associated with skin, hair and eye color, facial forms, height, and baldness. Even though, in some cases these information may not be taken into account as definitive forensic evidence, they can be very leading for an investigation. Forensic DNA Phenotyping is mostly used for investigations which has no known suspects or in missing person cases.
Color difference between individuals has a pattern just like the pigmentation of hair or skin. The amount of melanin and number of melanosomes in the outer layer of iris define the color difference. One of the first phenotyping tools developed and validated was the Irisplex System8 consisting of six SNPs distributed among pigmentation genes (HERC2, OCA2, SLC24A4, SLC45A2, TYR, and IRF4) (7). According to the studies, these tool accomplished to distinguish between brown and blue eye colors with more than 90 percent accuracy. This tool did not show high accuracy in Asian populations. This information is suggesting that it is necessary to conduct more studies that should be focused on different populations. Another disadvantage on this topic is intermediate eye colors. Prediction accuracy of these eye colors are lower when compared to the blue and brown eye colors.
Two types of melanin are responsible from the difference of hair color in people: Eumelanin and pheomelanin. If the amount of eumelanin is high, color of the hair tends to be darker and if the amount of eumelanin is low hair tends to be lighter. For example, in the case of red hair pheomelanin amount is higher than eumelanin. First model based on 22 SNPs made for prediction has the 81%–93% accuracy for each hair color category (7). A new system made in 2013 (HIrisplex System) , encompasses markers from MC1R, HERC2, OCA2, SLC45A2, KITLG, EXOC2, TYR, SLC24A4, IRF4, ASIP, and TYRP1 genes and, even though it has fewer markers than the model previously created by Branicki et al, it can reach similar accuracy values (75%–92%) (7).
Skin pigmentation is a complex pigmentation type since skin colors are responses to to the intensity of ultraviolet radiation in different regions of earth. This complexity is making the mapping studies hard for scientists. One of the major problems with skin color phenotyping is that it is not applicable for different populations across the planet. A global prediction model was created taking into account three (light, dark, dark–black) or five (very pale, pale, intermediate, dark, dark–black) skin tones, obtaining prediction accuracies ranging from 83%–97% for the three-category scale to 72%–97% for the five-category scale (7).