Genetic analysis

Genetic analysis of DNA: understanding DNA testing, polymorphisms and result reliability

DNA testing is a genetic analysis technique that allows a person to be identified (or multiple people to be compared) from a small quantity of biological sample.

The principle is simple:

• humans share a very large part of their DNA,

• but each individual also possesses a part of their DNA that is unique, forming a genetic fingerprint.

To understand how a laboratory establishes this genetic profile, we must return to the basics: what is DNA, how it is organised, and which regions are used to compare two profiles.

What is DNA? (simple definition)

DNA is a long molecule that contains the instructions necessary for the body to function.

In our cells, DNA is coiled and condensed in the nucleus in the form of "balls of wool" called chromosomes.

Each person has 46 chromosomes per cell:

• 23 inherited from the biological mother,

• 23 inherited from the father.

DNA manages a large part of our biological functions. It is the carrier of genetic information, and serves as a blueprint for the manufacture of many essential molecules.

Composition of DNA: nucleotides, amino acids and proteins

Nucleotides (A, C, G, T)

At the base level, DNA is made up of nucleotides. These are organic components (formed from chemical elements) that assemble into sequences.

There are 4 nucleotides, represented by:

• A (Adenine)

• C (Cytosine)

• G (Guanine)

• T (Thymine)

From DNA to proteins

In the body, the information encoded by DNA is used particularly to produce proteins.

In simplified terms:

• sequences of nucleotides allow more complex structures to be formed,

• these structures lead to the formation of amino acids,

• then amino acids assemble to form proteins.

Proteins have numerous roles: they participate in the creation of molecules, in the management of vital functions and in the transmission of information. Depending on their role, they may bear different names (enzymes, myosin, histones, etc.).

Genes: coding regions, non-coding regions, and the concept of locus

DNA in double helix and pairing rules

DNA is organised as a double helix: two long parallel and complementary strands of nucleotides, linked by molecular bonds.

The bases always pair rigorously:

• G is always opposite C (and vice versa),

• A is always opposite T (and vice versa).

This organisation makes DNA duplication easier and more reliable.

What is a gene?

A gene is a segment of DNA. Depending on its expression, it contributes to the cell's role (cardiac cell, liver cell, brain cell, etc.).

We generally distinguish:

• coding regions, used to produce new proteins,

• non-coding regions, which mainly play a regulatory role.

It is considered that non-coding regions represent approximately 98% of our DNA.

Locus: locating a precise region of DNA

Genes and certain sequences are positioned at precise locations on DNA. This position is stable in all human beings, which facilitates localisation.

When locating a precise region, we speak of a locus.

• In a coding region, the locus can be identified by the associated protein.

• In a non-coding region, the locus is often named according to a standard code.

Example: D18S52

• 18: chromosome 18

• S: unique sequence (in the sense of marker)

• 52: locus number

Polymorphisms: why each DNA is different

Even though we all belong to the same species, our diversity comes from small variations in DNA.

These variations are called polymorphisms. When comparing two randomly selected individuals, approximately 1 variation per 1,200 nucleotides is found.

Two types of polymorphisms (according to region)

There are two main cases:

• variation in coding region: the variation may affect the protein.

• variation in non-coding region: the variation is often observable by the number of repetitions of a sequence. We then speak of length polymorphism.

Length polymorphism and the concept of allele

Length polymorphism corresponds to a sequence repeated several times.

Example (repetitive sequence): AAGTA

• 11 repetitions in one person,

• 14 repetitions in another,

• 15 repetitions in a third.

The term used to designate a variant is allele.

During an analysis, a person generally has two alleles for a genetic characteristic:

• one allele from the paternal chromosome,

• one allele from the maternal chromosome.

Repetitive sequences: VNTR and STR (the most commonly used markers)

A repeated polymorphic sequence is classified according to its length:

• VNTR (Variable Number Tandem Repeats), or minisatellites: repetitions of sequences of at least 10 nucleotides.

• STR (Short Tandem Repeat), or microsatellites: repetitions of short sequences, of less than 10 nucleotides.

STRs have become established in genetic analysis for several reasons:

• they are very numerous (approximately 50,000 STR sequences in human DNA),

• they can be analysed in multiplex (several markers at the same time).

However, depending on the markers, they may sometimes present more limited polymorphism.

How is DNA analysis carried out in the laboratory?

The laboratory's objective is to obtain a usable profile, then to compare genetic markers to establish a genetic fingerprint.

1) Extraction and purification of DNA

The first step consists of extracting and purifying DNA.

The idea is to separate DNA from other substances that could hinder analysis. To do this, the sample is placed in a medium that eliminates external elements in order to preserve only the DNA molecule.

Depending on the type of analysis, sequences (often STRs) can be selected and cut with restriction enzymes.

Restriction enzymes are proteins (often of bacterial origin) capable of cutting DNA at specific sequences. They are widely used in genetic engineering and in laboratories.

2) Amplification by PCR (Polymerase Chain Reaction)

The second step is amplification by PCR. This technique allows, from a scant sample, the rapid copying of targeted DNA sequences in a large number of copies.

This is possible thanks to an enzyme: DNA polymerase, which reconstructs DNA from a previously separated helix.

The PCR mixture generally comprises:

• DNA sample (target sequence),

• additional nucleotides,

• primers (small strands complementary to the sequence to be copied),

• DNA polymerase.

The mixture is subjected to temperature cycles:

• denaturation (approximately 90°C): separation of the two strands,

• hybridisation (approximately 45°C): fixation of primers,

• elongation (approximately 72°C): reconstruction of the missing strand.

At each cycle, the number of copies doubles. In 30 to 40 cycles, millions of copies of the target sequence are obtained.

3) Electrophoresis: separation of fragments and profile reading

The third step is electrophoresis, which allows fragments to be separated according to their size under the effect of an electric field.

DNA being negatively charged, fragments migrate towards the positive pole.

• the smaller a fragment, the more rapidly and far it migrates,

• fragments of the same size form identifiable bands.

Thanks to this reading, the laboratory can determine the composition of the fragment: the number of nucleotides and the number of repetitions.

The set of results forms a person's genetic fingerprint, which can then be compared to determine a filiation link.

With the exception of true twins, the probability that two people have the same genetic fingerprint is extremely low (approximately 1 in 3 billion).

Analysis of mitochondrial DNA (mtDNA): a non-standard test

The mitochondrial DNA (mtDNA) test is a genetic analysis that does not rely on nuclear DNA (that contained in the nucleus), but on DNA present in mitochondria.

Mitochondria: what are they for?

Mitochondria are specialised structures (organelles) that produce energy for the cell.

It is considered that they originate from ancient bacteria that entered into symbiosis with a cell several million years ago.

Why is mtDNA particular?

Mitochondrial DNA:

• is circular,

• is present in very numerous copies (several hundred mitochondria per cell, with several DNA copies each),

• can be isolated from old or degraded samples, where nuclear DNA is not detectable.

Maternal transmission of mtDNA

Mitochondrial DNA is transmitted through the maternal line: during fertilisation, the ovum provides the mitochondria.

Thus, members of a sibship generally share the same mitochondrial DNA transmitted by the mother, she herself having inherited it from her mother, and so on through the maternal line.

MtDNA does not always identify an individual 100% (several people may share identical mtDNA), but it can be useful for:

• verifying a kinship link,

• exploring certain origins,

• analysing degraded samples.

Mutations sometimes appear over generations. Their accumulation explains why distinct maternal lines eventually present different mtDNA.

What is the reliability of a DNA test?

The reliability of a DNA test depends on several factors.

1) Laboratory accreditation

Checking the laboratory's accreditation ensures the analysis methods used and the seriousness of quality control.

Accreditation corresponds to an international standard that the laboratory can obtain after verification of the process by an external body.

It may also be important if you are seeking a level of legal recognition depending on the context.

2) Declaration of your situation

Before ordering, clearly describe the family situation, doubts and possible relationships between participants.

The result also depends on proper understanding of the context, as interpretation relies on probability calculations.

3) Test type

Not all DNA tests are equal depending on the situation.

As a general rule, it is advisable to carry out the test with the person directly concerned, as this increases the relevance and reliability of the comparison.

4) Sample type

Reliability does not depend solely on sample type, but not all samples always reliably provide sufficient genetic information.

A properly collected and stored sample greatly facilitates analysis.