DNA testing is a genetic analysis technique that makes it possible to identify a person from a small amount of biological samples.
This analysis therefore makes it possible to define a genetic identification fingerprint which is based on the following facts:
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Humans have a large majority of DNA in common
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Each individual has in his DNA a part that is unique
To understand how the lab shines a light on our genetic profile, you have to start from the ground up and understand what exactly DNA is.
DNA is a long molecule of information that is rolled up and condensed into a ball present in our cell nuclei. A person has in each of these cells 46 balls of DNA that we call chromosome: 23 inherited from our biological mother and 23 from our father.
It is important to note that DNA manages almost all of our biological functions ; it is the carrier of our genetic information and also provides manufacturing instructions.
Composition of DNA
DNA is a molecule fundamentally composed of nucleotides. Without delving into too much detail, nucleotides are organic components made up of chemical elements. There are four types of nucleotides, represented by the letters A, C, G, and T: Adenine, Cytosine, Guanine, and Thymine.
In our DNA, nucleotides group together into more complex structures to form what we call amino acids. Moreover, these amino acids combine further to eventually create the proteins in our body.
Proteins perform a multitude of functions within the organism. They regulate our entire biological activity, from molecule creation to vital function management and information transmission. Depending on their roles, proteins can have different names, such as enzymes, myosin, or histones.
DNA Genes
DNA is therefore a complex structure that brings together different chemical components in a double helix organization. Two long parallel and complementary strands of nucleotides linked together by molecular bonds.
Know that in DNA, all its architecture and the relationship between nucleotides are rigorously hierarchized by chemical elements:
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In front of a G, there is always a C and vice-versa
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In front of an A, there is always a T and vice versa
This rigorous organization allows the molecule to be duplicated very easily.
A gene is a strand in the DNA molecule . It is a segment of DNA which, depending on its expression, defines the role of its cell. (heart cell, liver cell or brain cell...)
The role of the gene according to its cell thus determines the existence of two major functions on the DNA molecule:
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The coding regions, which are used to produce and create new proteins
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The non-coding areas, which have more of a role in protein regulation
It is considered that the non-coding areas cover about 98% of our DNA.
Still with the same logic, the genes are just as rigorously positioned in a specific way on the DNA. This makes it easier to locate a particular gene, because its position remains unchanged in all human beings.
When a specific gene is located, it is then called a locus .
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For a coding DNA area , the locus is identified with the name of the protein used.
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For a non-coding DNA zone , each locus is listed according to a specific code:
The D18S52 locus: is located on chromosome 18, it supports a nucleotide sequence that is not found elsewhere and it bears the number 52, i.e. D18S52 .
Polymorphisms
Of course, human beings are all part of the same species with a huge number of similarities in nucleotide structure. However, our extraordinary diversity is rooted in minute variations .
These variations between individuals are called polymorphisms and the analysis of a DNA sample allows us to observe these variations and compare them. By taking two individuals at random, we find about 1 variation every 1200 nucleotides.
There are two types of variations depending on the coding or non-coding region of DNA.
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When the polymorphic variation is in a coding region, the variation is visible on the protein of the locus (structure of the nucleotide in amino acid, and the amino acids in protein)
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When the polymorphic variation is in a non-coding area, the variation is visible by the number of nucleotide repeats. This is called length polymorphism.
Length polymorphisms are repetitive sequences of nucleotides which can be repeated several times in a row like the group: AAGTA which can vary from one person to another and thus be repeated 11 times in one person, 14 times in a second person or 15 times at a third.
The term used to refer to the variants between individuals is “ Allele ”.
During an analysis, an individual will each have two alleles for each genetic trait. One allele representing the variant present on the paternal chromosome and one allele representing the variant present on the maternal chromosome.
A repeating polymorphism sequence is composed of a minimum of 10 nucleotides. For this, it is commonly called VNTR (Variable Number Tandem Repeats) or minisatellite .
A polymorphism sequence that repeats with a small number of nucleotides (less than 10), we then speak of STR (Short Tandem Repeat) or microsatellite .
Short STR-like sequences have finally established themselves in genetic analysis because of their advantages:
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they are numerous (about 50,000 sequences of this type in human DNA)
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they can be analyzed simultaneously (multiplex analysis)
On the other hand, they sometimes suffer from limited polymorphism.
Repetitive Sequences: VNTR and STR
The first step in genetic analysis is DNA extraction and purification . To do this, you have to somehow unhook the DNA molecule from its support and dissolve the substances that could interfere with the progress of the analysis. The scientists then immerse the sample in an aqueous medium which will eliminate all the external substances to finally retain only the DNA molecule.
Depending on the type of analysis, the STR sequences are chosen and carefully cut with a natural protein: the restriction enzyme .
Restriction enzymes are particular proteins, because originating from bacteria, they have the ability to cut DNA molecules at specific sequences depending on the enzyme chosen. They are therefore widely used tools in genetic engineering and in biology laboratories.
DNA extraction
The second step is PCR amplification . It is a method allowing from a scanty sample to quickly copy precise DNA sequences in very many copies . This copying technique which makes it possible to double the amount of DNA in a very short time is possible thanks to the discovery of the enzyme named " DNA polymerase "(protein) which allows the perfect reconstruction of a previously separated DNA helix.
PCR amplification is done with the mixture of the following active ingredients:
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DNA sample : previously cut segment
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Additional nucleotides
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DNA primers : single strands complementary to the sample to be copied
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DNA polymerase : enzyme which recognizes primers and assembles nucleotides to copy the target DNA
The mixture is subjected to rapid temperature variations in a programmed cycle.
Each cycle consists of 3 steps:
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90° denaturation to separate DNA helices into 2
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45° hybridization to attach primers to DNA fragments
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72° elongation which allows the reconstruction of missing DNA by the DNA polymerase enzyme using primers and added nucleotides
At each cycle, the number of copies is doubled . In 30 or 40 cycles, millions of copies of the target sequence are obtained.
PCR (polymerase chain reaction)
The third step is the separation of DNA by electrophoresis . This method makes it possible , under the effect of an electric field, to separate the proteins from the DNA according to their size and their molecular weight . The experiment can be done in a tube or in a gel, and allows the observation of the migration of the DNA sequence according to its composition.
The fragments move according to their size towards the positive pole, because the DNA is negatively charged. The smaller the fragment, the faster (and therefore farther) it migrates. All the fragments of the same size form a recognizable line and make it possible to characterize their DNA content.
Thus, depending on the result of the migration after electrophoresis, the laboratory is able to determine the composition of the fragment: the number of nucleotides and its number of repetitions
The analysis of the fragments obtained by electrophoresis forms the genetic fingerprint of a person which can then be compared to determine a parentage link. Since the length of a particular fragment can vary from one individual to another, with the exception of identical twins, the probability that two people have the same genetic fingerprint is almost zero (1 in 3 billion).
Electrophoresis
Mitochondrial DNA Analysis
The mitochondrial DNA (mtDNA) test is a genetic analysis that does not use the genetic information that is in our DNA. It is a non-standard test, which is not oriented on the nuclear DNA present in our cells as previously, but rather on the analysis of the DNA of the mitochondria.
Mitochondria are ancient bacteria that entered a cell to form a symbiotic relationship, millions of years ago. This relationship has transformed the bacterium into a real organelle for the cell.
The organelles are small compartments specialized in certain functions that regulate the life and activity of the cell. The mitochondria is specialized today in the production of energy for the cell.
Mitochondrial DNA therefore corresponds to the DNA found inside the mitochondria, hence its name, and not to the DNA present in the nucleus of the cell which is the support of the genetic heritage of an individual.
Mitochondrial DNA is circular DNA that has several hundred mitochondria per cell and each contains about ten copies of its DNA. Thus it will therefore be present in several thousand copies whereas nuclear DNA is only present in two copies. For this reason, mitochondrial DNA can be isolated from old or very degraded samples where nuclear DNA is not detected.
Mitochondria
Mitochondrial DNA (mtDNA) analysis cannot identify an individual with 100% certainty, as multiple people can share the same mtDNA. However, it is highly useful for verifying maternal relationships or tracing origins.
This is because mtDNA is passed down exclusively through the maternal line. During fertilization, when the maternal ovum and paternal sperm fuse, only the ovum contains mitochondria, allowing for the transmission of mtDNA.
As a result, all siblings inherit the same mitochondrial DNA from their mother, who in turn inherited it from her mother, continuing along the maternal lineage.
One might assume that all humans would have identical mitochondrial DNA since it is passed unchanged through generations along the maternal line. While it is possible that our species once shared the same mtDNA sequence, natural mutations occasionally occur. When these mutations happen in reproductive cells, they may be passed on to descendants.
Over the course of human evolution, these accumulated mutations have created distinct mtDNA sequences for descendants of each unrelated family (unless they share a maternal ancestor).
Unlike nuclear DNA, mitochondrial DNA does not contain repetitive sequences, and inter-individual variations are sometimes visible in a single nucleotide. Thus, mitochondrial DNA polymorphism is structural rather than repetitive, as seen in nuclear DNA.
The Analysis Process
Mitochondrial DNA analysis focuses on these polymorphisms within a non-coding region called the control region.
The sequences are amplified using PCR (Polymerase Chain Reaction) and then detailed through sequencing. This process provides the complete sequence of nucleotides, allowing researchers to identify variations.
Sequencing is a meticulous process since it involves mapping the entire mtDNA region to identify variations, which can sometimes occur in just a single nucleotide. On average, around 8 nucleotide differences are found among the 600 analyzed.
Mitochondrial DNA Inheritance
How reliable is a DNA test?
The reliability of the results of a DNA test will depend on several factors:
1. Laboratory accreditation
Checking the laboratory's accreditation allows you to be sure of the analytical methods that scientists use when looking for a parentage link. An accreditation is an international standard that a laboratory can acquire after verification of the entire process by an external committee.
An accreditation gives the laboratory the possibility of genetic analyses which may be legally accepted.
2. The declaration of your situation
Be sure to communicate well the family situation before your order, your doubts and the possible relationships between the participants. The result of the DNA test will depend on your declaration, because it is linked to a deduction of possibility.
3. The type of test
All DNA tests are not equal on the probability ratio they offer, and depending on the basic situation several tests are possible and some more reliable than others. As a general rule, it is always advisable to do DNA tests directly with the person concerned.
4. Type of sample
Reliability of results does not depend on sample type, but not all samples reliably provide enough genetic information to do a DNA test.
