Genetic fingerprint
Genetic fingerprinting (also called DNA testing or DNA analysis) is a technique used to distinguish between individuals of the same species using samples of their DNA. Its invention is due to Dr. Alec Jeffreys, of the University of Leicester, who unveiled his new technique in 1984. The first practical result in forensic medicine served to convict Colin Pitchfork of the Narborough murders in 1983 and of Enderby in 1986.
The technique is based on the fact that two human beings have a large part of their DNA sequence in common and to distinguish between two individuals, the repetition of highly variable sequences called minisatellites or VNTRs can be exploited. It will be unlikely that two unrelated humans will have the same number of minisatellites at a given locus. In SSR/STR profiling (which is distinct from imprinting) polymerase chain reaction (PCR) is used to obtain enough DNA to detect the number of repeats at various loci. It is possible to establish a selection that is very unlikely to have arisen by chance, except in the case of identical twins, who will have identical genetic profiles.
Genetic fingerprinting is used in forensic medicine to identify suspects from blood, hair, saliva, or semen samples. It has also led to several exonerations of convicted persons. It is also used in applications such as the identification of human remains, paternity tests, compatibility in organ donation, the study of populations of wild animals, and the establishment of the origin or composition of food. It has also been used to generate hypotheses about the migrations of human beings in prehistory.
Microsatellites show greater variation than the rest of the genome since they contain sequences in different repetitions and with different degrees of recombination due to locus instability.
Reference samples
To identify the DNA, a DNA extraction must be carried out, which can come from samples such as:
- Personal items (e.g. toothbrush, shaving machine).
- Sample bank (such as a semen bank or a tissue biopsy).
- Relatives.
- Human remains previously identified.
Some reference samples are collected with a buccal swab.
Genetic fingerprinting#34;identification techniques
These variations can be detected with the following techniques:
RFLP Analysis
The analysis consists of making a Southern assay (or Southern blot, a method used to verify whether or not a certain DNA sequence is present in a DNA sample analyzed) and using specific probes to detect VNTRs (variable number of tandem repeats).
First, the DNA to be analyzed is separated from other materials. Next, it must be cut into fragments of different sizes using restriction enzymes, which are proteins that cut DNA without damaging the bases. Fragments are ordered by size using gel electrophoresis. DNA, which is negatively charged, moves forward in the electric field. Smaller molecules move faster through the gel, so they will be located farther from the origin than larger fragments. Then by heat or alkaline solution, gel is applied in order to denature the DNA and it is separated into individual fragments. Once this is done the DNA is now ready to be analyzed using a radioactive hybridization reaction probe.
To make the probe radioactive, DNA polymerase is needed. The DNA to be subjected to radioactivity is placed in a test tube. The polymerase is then added to the tube. It dissolves and waits for it to start working. As the DNA polymerase patches break the DNA, the current ones are replaced by the new nucleotides in the tube. Every time the sample has a guanine base, the cytosine will be made radioactive. In the DNA repeat, the polymerase also becomes radioactive. The radioactive parts are ready for use. Now the radioactive probe can be used to create a hybridization reaction. Hybridization occurs when two genetic sequences are joined by the hydrogen found in the base pairs. There are two of these between adenine (A) or thymine (T) and three of cytosine (C) or guanine (G). To perform hybridization, the DNA must be denatured.
The radioactive denatured DNA and probe should be placed in a plastic bag filled with saline and sealed tightly. The probe will adhere to denaturing DNA wherever it is appropriately located. The probe and the DNA do not have to match exactly. This process ends up making a DNA pattern of the fingerprints. Every person has a VNTR that he inherited from one of her parents, and VNTRs are unique to each person.
PCR analysis
It consists of applying the PCR technique to amplify specific regions with the appropriate primers. With the invention of the polymerase chain reaction (PCR), genetic technology had an important advance in the ability to recover information from very small samples. PCR consists of the amplification of specific regions of DNA using temperature and a thermostable polymerase enzyme together with fluorescence labeling in a specific DNA sequence. There are commercial kits that use discriminating single nucleotide polymorphisms (SNPs) available, these kits use PCR used to amplify specific regions with known variations and hybridization with tether probes on cards, resulting in a colored spot corresponding to a particular order.
The main criticisms of the RFLP method concern its slowness and the large amounts of DNA it requires to obtain useful results. This led to PCR-based methods that require smaller amounts of DNA that can also be more degraded than those used in RFLP analyses.
PCR assays are extremely valuable for identifying new members of a gene family.
AmpFLP
This is a technique for amplifying polymorphic regions (with many polymorphisms [6]), preferably the D1S80 locus. This analysis can be automated, and allows for the easy creation of phylogenetic trees based on the comparison of individual DNA samples.
Based on variable number of tandem repeats (VTNR) to distinguish various allelic polymorphisms, which are separated on a polyacrylamide gel used in an allelic ladder (as opposed to molecular weight). Bands could be visualized by silver gel staining. A popular location for fingerprinting is the D1S80 locus. Like PCR-based methods, degraded DNA or very small amounts of DNA can cause allelic dropout.
Due to its relatively low cost and ease of startup and operation, AmpFLP remains popular in low-income countries.
STR (Short Tandem Repeat) Analysis
It consists of the amplification of sequences with small tandem repeats that are not conserved within the species. Once amplified, the fragments are separated to check the number of repetitions (by capillary or gel electrophoresis), so that patterns of repetitions can be distinguished that can be comparable and associable.
This method uses highly polymorphic regions that have short DNA repeat sequences (the most common is a 4 base repeat, but other lengths are in use, including 3 and 5 bases). Because different people have different numbers of repeat units, these regions of the DNA can be used to differentiate between people. These STR loci (sites) are attacked with sequence-specific primers and amplified by PCR. The resulting DNA fragments are then detected and separated by electrophoresis. There are two methods of separation and detection, capillary electrophoresis (CE) and gel electrophoresis.
The STR polymorphisms shown in each region are inherently very common, typically each polymorphism is shared by about 5 - 20% of people. When looking at multiple loci, it is the unique combination of these polymorphisms in an individual that makes this method of discrimination an identification tool. The STR regions that are tested in a person become a test discrimination. Therefore, if the DNA profiles of two different samples (for example, in the analysis of suspects in a crime or in a paternity test) do not match, the possibility of a relationship between these two samples is directly excluded and no statistical analysis is performed. later. However, if both profiles match, a subsequent statistical analysis must be performed to see the probability that the sample of evidence comes from a random person in the population. When interpreting the information coming from a test in which the DNA profile of the suspect in a crime matches the profile of the sample taken at the crime scene, it is crucial to express that the suspect is many times more likely to be the culprit than any other random person.
From one country to another there are different STR systems in use for DNA analysis. In North America systems that amplify the CODIS 13 core loci are nearly universal, while in the UK the SGM+ system which is compatible with the National DNA Database is in use. Whichever system is used, many of the STR regions used in the test are the same. These DNA analysis systems are based around multiplex reactions, in which many STR regions will be tested at the same time.
Capillary electrophoresis (movement by applying an electric field), allows the injection of DNA fragments into a thin glass tube (capillary) filled with polymers. The DNA is drawn through the tube by the application of an electric field, the separation of the fragments occurs in such a way that the smallest fragments travel faster through the capillaries. The fragments are then detected using fluorescent dyes that are attached to the primers used in the PCR. This allows multiple amplified chunks to run simultaneously, known as multiplexing. Sizes are then assigned using DNA size as labeling rules that are added to each sample, and the number of repeats are determined by comparing the size of the allelic ladder, which contains a sample of all common sizes of possible repeats. Although this method is expensive, with larger machine capacity and higher throughput being used to reduce cost/sample it is possible to reduce delays that exist at many government crime facilities.
Gel electrophoresis is one of the procedures that is used using similar principles known as 'CE', but instead of using a capillary, the large polyacrylamide gel is used to separate the DNA fragments. An electric field is applied, as in CE, but instead of running all the samples through a detector, the smallest fragments are run near the bottom of the gel and the entire gel is scanned and saved on a computer. This produces an image that shows all of the bands corresponding to different sizes and repeats the allelic ladder. This method does not require the use of size standards, since the allelic ladder is run alongside the samples and serves this purpose. Visualization can be through the use of labeled fluorescent dye primers in the silver stain or by gel prior to scanning. Although it is cost-effective and can be quite high-yielding, applying ITS silver staining is discontinued. Additionally, many labs are phasing out gels in favor of EC, making the cost of the machines more manageable.
The true power of STR analysis lies in the statistical power of discrimination. In the US, there are 13 core loci (DNA sites) currently used for discrimination in CODIS. Because these loci use a variety independently (having a given number of repeats at one locus does not change the probability of having any number of repeats at any other locus), the product odds rule can be applied. This means that if someone has DNA of type ABC, in which the three loci are independent, we can say that the probability of having that type of DNA is the probability of having type A times the probability of having type B times is the probability of having type C. The most prevalent DNA fingerprinting method used today is PCR-based and uses short tandem repeats (STR). This method uses highly polymorphic regions that have repeated short DNA sequences (the most common is a 4 base repeat, but other lengths are in use, including 3 and 5 bases). Because different people have different numbers of repeat units, these regions of the DNA can be used to discriminate between people. These STR loci (sites) are attacked with sequence-specific primers and amplified by PCR. The resulting DNA fragments are then detected and separated by electrophoresis. There are two methods of separation and detection, capillary electrophoresis (CE) and gel electrophoresis.
The STR polymorphisms displayed in each region are inherently very common, typically each polymorphism will be shared by about 5 - 20% of people. When looking at multiple loci, the unique combinations of these polymorphisms in an individual make this method of discrimination an identification tool. The other STR regions that are tested in a person become a test discrimination.
Y chromosome analysis
Specific primers have been created for regions of the Y chromosome that amplify sequences only inherited from the paternal side. Therefore, this type of analysis only serves to compare relatives on the father's side and males.
Mitochondrial Analysis
It is used in highly degraded samples, since mitochondrial DNA has more copies than nuclear DNA. The HV1 and HV2 regions are amplified and are always compared with relatives on the maternal side, since mitochondria are exclusively inherited from the mother. Forensic scientists amplify the HV1 and HV2 region of the mtDNA and then each region is sequenced to a single nucleotide and the differences compared to the reference.
Practical applications of genetic fingerprinting
- Forensic science. Compare suspects with blood, hair, saliva or debit semen samples.
- Paternity Tests
- Study compatibility in organ donations.
- Studies of evolution of wild animal populations.
- Generation of hypothesis on human migrations in history.
- In children adopted or conceived by assisted reproduction techniques using donated gametes. In these cases, children do not share the genetic code with their parents, so that in biological identification, DNA studies cannot be used as compared to parents.
Examples of use of the “genetic fingerprint”
One of the first cases of international repercussion was that of Josef Mengele, a Nazi war criminal, whose supposed remains were discovered in 1985 in a Brazilian cemetery. In 1988 DNA extracted from a skeletal bone was compared with DNA from the blood of Mengele's wife and son. The conclusion, with a 99.94% probability, was positive for the identification of the remains found as belonging to Mengele.
The acceptance of genetic fingerprinting as a forensic method, in criminal cases, took much longer. Convicting a person on this basis was for years considered too risky, despite the fact that its comparison with other less precise methods, such as visual identification, benefited him. However, refinement and standardization of the method have led to its universal acceptance. In recent cases, people sentenced to life imprisonment and the death penalty at the time have been exonerated, based on their DNA. The first case was that of the American Kirk Bloodsworth, sentenced to death in 1985 for the murder and rape of a nine-year-old girl. The review of the case occurred in 1992 with the result that Bloodsworth was released in 1993. However, the use of genetic fingerprinting in these cases can lead to problems for various reasons, some of the most relevant being the following:
- Sometimes we can find DNA mixtures at the crime scene or DNA is damaged, which can lead to false results.
- In certain countries, the DNA of people who are arrested (sometimes even if they are not found guilty), is incorporated into a database, which can lead to ethical problems because it violates the presumption of innocence.
- Today technology is sufficiently developed to make it possible to create a synthetic DNA, which could be left at the crime scene with the aim of inculcating someone from it.
One of the most famous cases of genetic fingerprinting was Dolly the Sheep, presented in 1997 as the first mammal cloned from an adult cell. This statement could not be scientifically supported until it was verified that their DNA was identical to that of the donor sheep. It was Jeffreys' team that proved "beyond any reasonable doubt that Dolly comes from a mammary tissue cell taken from the adult donor sheep," Esther Signer, author of the analysis, explained at the time.
In certain countries, this type of evidence is voluntary except in the case of legal order, since it could be used as decisive evidence in trials if the judge in a case deems it appropriate.
The next step is taken by some countries, including the US and the UK, creating databases with the genetic profiles of many of their inhabitants that would facilitate the resolution of criminal cases by comparing profiles.
Currently, Alec J. Jeffreys continues to study the genetic fingerprinting technique by conducting recent studies. Specifically, in 2006 a study was published that studied how a single strand of DNA can provide information on ectopic recombinations, revealing the different meiotic recombination pathways in the α-globin cluster and its relationship with population drift. And later, in 2007, the study of highly polymorphic minisatellite regions (VNRT's) in gene flow through the evolution of mice was published.