DNA analysis techniques

Approximately 99.9% of the genome, or 2.97 billion base pairs, is shared between unrelated humans. Within the remaining 0.1% lies the information that makes each human being unique. Analysis of each individual’s genome provides us with unique and personalised information about him. For this reason, it is very important the development of techniques that allow us to accurately read the genetic information of each individual.

These techniques are mainly used in clinical practice, for example for the diagnosis of pathologies, determination of different stages in the case of patients affected by cancer or the adequacy of treatment for patients with certain pathologies.

What types of DNA profiling techniques are there?

There are numerous forms of analysing the DNA, therefore, there are numerous types of techniques. Some of the current techniques and their specific functions are detailed below:

Karyotype with G bands

The karyotype is a technique that allows the observation of the shape of the chromosomes (bundles in which DNA is organised) by staining with a special stain (Giemsa). By means of this staining it is possible to observe the number of chromosomes present in a cell, which chromosomes are present and whether their internal organisation is correct. This technique is used to diagnose patients with changes in the number of chromosomes, as in the case of people affected by Down’s syndrome whose DNA has 3 chromosomes 21. It is also the technique used to see reorganisations between chromosomes, as in the case of patients affected by leukaemia.

Sanger Sequencing

A sequencing technique developed by Frederick Sanger in 1977. Two years after its invention, it allowed the sequencing of the first whole genome of a bacteriophage.

This technique is based on a cyclical process: extraction, denaturation, elongation and detection. In this technique, only one part of the DNA is used as a template per reaction and a signal is emitted depending on the type of marking that each basic unit of the DNA has (A, T, C, G), which allows the distinction between each base of the sample used as a template. In this way, with different reactions for the same sequence, we will be able to collect all the information from it, but the number of reactions that will occur at the same time is limited.

This technique is still used today for the analysis of small regions as its reliability in these cases is very high.

Next Generation Sequencing (NGS)

The human genome consists of 3 billion base pairs, which are chemical molecules whose organisation into genes explains the functioning of the organism. Deciphering our genome took about 13 years and 2700 million dollars as part of the human genome project. Today, costs and time have been radically reduced thanks to new sequencing techniques (NGS). This set of new techniques subsequent to Sanger’s development, together with the advance of bioinformatics tools that analyse the results of the technique, allow a greater volume of data to be read at a lower cost.

With massive sequencing techniques (or Next Generation Sequencing), the number of reactions that can be performed at the same time increases, thus producing a more complete reading of the genome in a shorter time. One of the few disadvantages of these new methods is their lower precision compared to the Sanger method, although advances in bioinformatics will allow this disadvantage to disappear.

Multiplex Ligation-dependent Probe Amplification (MLPA).

This technique is used for the detection of copy number changes (generally 2 copies per region) in regions of interest in genes. Although the vast majority of inherited genetic diseases are due to point mutations, i.e., DNA sequence changes detectable by Sanger sequencing or NGS, it has been reported that 5% of genetic diseases are caused by copy number changes in DNA regions.

For example, 75% of the variants causing Duchenne Muscular Dystrophy (an X-linked inherited disease) are deletions or duplications of large regions of the DMD gene.

Although it is a high-throughput method, it also has a number of limitations such as a high sensitivity to contamination or the inability to analyse a single cell.

Array Comparative Genomic Hybridization (aCGH)

This technique allows the detection of alterations in the copy number of several genes simultaneously. It is based on competitive ligation between the DNA of the person being analysed and the reference DNA to specific regions of a plaque. Both types of DNA will be labelled and the intensity of the sample compared to the reference sample will determine the amount of genetic material of the person being tested in a given region.

This type of test makes possible to study microdeletions or microduplications that would go undetected in other DNA tests. However, aCGH, although it analyses changes that affect large regions of the genome, does not allow the detection of changes in the positions of DNA regions.

Triplet repeats primed polymerase chain reaction (TP-PCR)

TP-PCR allows the identification of tandem repeat expansions of certain combinations of the bases that make up DNA in patients with diseases caused by an increase in these repeats, such as patients affected by Huntington’s disease, Fragile X syndrome or Friedreich’s ataxia. This technique makes possible to count the number of repeats in the combination and, to date, remains the only sufficiently reliable technique for diagnosing this type of rare diseases.