RECOMBINANT DNA: TECHNIQUE, APPLICATIONS AND FUNDAMENTALS - BIOLOGY - 2025

The recombinant DNA (rDNA or rDNA) is an artificial nucleic acid molecule created in the laboratory, by integrating two segments of interest organizations. It is also known as chimeric DNA, thanks to its hybrid property. This type of DNA is not found in nature.

The basic methodology to generate it includes: (a) the selection of a target DNA, and its insertion into another DNA fragment (generally a bacterial plasmid); (b) the introduction of this plasmid into a bacterium, (c) the selection of the bacteria by means of antibiotics and finally (d) the expression of the gene.

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The technique takes advantage of a set of enzymes that make it possible to copy and paste specific DNA fragments according to the researcher's judgment.

The goal of recombinant technology is, in most cases, the expression of a protein (known as a recombinant protein) desired by the molecular biologist for future research or to create a protein of commercial and therapeutic value - such as human insulin, for example.

Basics of the recombinant DNA technique and its use in genetic engineering

The central dogma of molecular biology

All organic beings that we know share several characteristics. One of them is the nature of the genetic material and the way proteins are made - a process known as the central “dogma” of molecular biology.

With the exception of a couple of viruses, all organisms store genetic information in DNA (deoxyribonucleic acid), collected in a very compact and organized way in the nucleus of the cell.

For gene expression, the DNA molecule is transcribed into messenger RNA, and the latter is translated into the language of amino acids, the building blocks of proteins.

What is a recombinant DNA?

Between the 1970s and 1980s, molecular biologists began to take advantage of the processes that naturally occur inside the cell and were able to extrapolate them to the laboratory.

In this way, a gene of animal origin (a vertebrate, for example) could be inserted into a segment of DNA from a bacterium; or the DNA of a bacterium could be combined with a viral DNA. Thus, we can define a recombinant DNA as a molecule made up of DNA from two different organisms.

Once this hybrid or recombinant molecule has been created, the gene of interest is expressed. With the word expression we want to refer to the process of translation to protein.

Restriction enzymes and ligases: the key to the process

A key element in developing recombinant DNA technology was the discovery of restriction enzymes.

These are protein molecules that exhibit the ability to cleave DNA (nucleases) into specific sequences, serving as “molecular scissors”. The fragments generated by these enzymes are called restriction fragments.

Said enzymes can produce symmetric cuts in the target sequence (in both chains at the same height) or asymmetric cuts. A key aspect of the action of restriction enzymes is that after the cleavage of the chains, a "loose edge" is obtained, complementary to the other edge cut by the same enzyme.

Some examples are ECOR 1 and Sma 1. Currently more than 200 types of restriction enzymes are known and commercially available.

To be useful, a scissors must be accompanied by glue. This sealing action of the DNA (previously treated with restriction enzymes) is carried out by ligases.

Technique: how is the DNA of an organism artificially modified in the laboratory?

Below we will describe the main steps that recombinant DNA technology requires. All are carried out by professionals in a molecular biology laboratory.

What is a "clone"?

Before continuing with the experimental protocol, we must note that in molecular biology and biotechnology the term "clone" and the verb "clone" are widely used. This could lead to confusion.

In this context, we are not referring to the cloning of an entire organism (as in the case of the famous Dolly the sheep, for example), but to the cloning of a piece of DNA, which can be a gene. That is, producing many copies - genetically identical - of the sequence.

1. Isolation and obtaining of DNA

The first step is to decide which sequence you want to use. This depends entirely on the researcher and the objectives of her work. This DNA must then be isolated and purified. The methods and procedures to achieve this depend in turn on the body and the tissue.

Generally, a piece of tissue is taken and subjected to treatment in a lysis buffer with proteinase K (a proteolytic enzyme) and then the DNA is extracted. Subsequently, the genetic material is fragmented into small fragments.

2. Cloning vector

After the preparatory steps, the researcher seeks to introduce the DNA segment of interest into a cloning vector. From now on we will call this segment of DNA white DNA.

Plasmids

One of the most used vectors in a plasmid of bacterial origin. A plasmid is a double-stranded, circular DNA molecule that is found naturally in bacteria. They are foreign to the bacterial chromosome - that is, they are extrachromosomal, and are found naturally in these prokaryotes.

The basic elements of a vector are: (a) an origin of replication, which allows DNA synthesis; (b) selection agent, which makes it possible to identify the organisms that carry the plasmid with the target DNA, such as resistance to some antibiotic; and (c) multicloning site, where the sequences that will be recognized by the restriction enzymes are found.

The first successful recombinant DNA in the laboratory was cloned into the plasmid pSC101 from the bacterium E. coli. This contains a restriction site for the restriction enzyme EcoRI and a gene for resistance to an antibiotic, in addition to the origin of replication.

The insertion of the target DNA in the plasmid is carried out using the molecular tools of restriction enzymes and ligases described in the previous section.

Remaining vector types

In addition to plasmids, DNA can be inserted into other vector, such as bacteriophage lambda, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and phagemids.

3. Introduction of recombinant DNA

Once the recombinant DNA molecule (gene of interest in the plasmid or other vector) has been obtained, it is introduced into a host or host organism, which can be a bacterium.

To introduce foreign DNA into a bacterium, a technique called bacterial transformation is used, where the organism is subjected to a treatment with divalent cations that makes it susceptible to taking up DNA.

Methodologically, we cannot guarantee that 100% of the bacteria in our culture have effectively taken up our recombinant DNA molecule. This is where the portion of the plasmid that contains antibiotic resistance comes into play.

Thus, the bacteria that have taken up the plasmid will be resistant to a certain antibiotic. To select them, it will be enough to apply said antibiotic and take the survivors.

4. "Harvest" the protein

After selecting the bacteria with our recombinant DNA, we proceed to use the host's enzymatic machinery to generate the protein product of interest. As the bacteria reproduce, the plasmid is passed on to their offspring, so it is not lost during division.

This procedure uses the bacteria as a kind of protein "factory". Later we will see that it has been a very relevant procedure in the development of effective medical treatments.

Once the culture is ready and the bacteria have produced large amounts of protein, the cell is lysed or disrupted. There is a wide range of biochemical techniques that allow the purification of proteins according to their physicochemical characteristics.

In another experimental context, we may not be interested in generating the protein, but rather we are interested in obtaining the DNA sequence per se. If this were the case, the plasmid would be used to create multiple copies of the fragment of interest in order to have enough of the target DNA to carry out the relevant experiments.

Applications

Recombinant DNA technology opened an infinite number of possibilities in molecular biology, biotechnology, medicine, and other related areas. Its most outstanding applications are the following.

Genetic analysis

The first application is directly related to molecular biology laboratories. Recombinant DNA technology enables researchers to understand the normal function of genes, and the generated proteins can be used in further research.

Pharmaceutical industry

Proteins produced using the recombinant DNA procedure have applications in medicine. Two very relevant examples in the field are human insulin and growth hormone, which is applied in patients who lack this protein.

Thanks to recombinant DNA, these proteins can be generated without the need to extract them from another human being, which represents additional methodological complications and health risks. This has helped improve the quality of life for countless patients.

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