Recombinant DNA technology

First, the gene of interest is identified and located in the DNA. The restriction enzymes are responsible for cutting segments of the desired gene.

The selected fragment is linked to the transfer vector (a bacterial plasmid) with the help of DNA ligase enzymes, obtaining a hybrid or recombinant DNA fragment.

This DNA molecule is transferred to the host cell, where the recombined DNA is replicated and transmitted to daughter cells. Thus, a clone of cells has been created that contain the gene from a different cell.

With this technique, it has been achieved, for example, that bacteria have the human genes necessary to synthesize insulin, or that mice produce human growth hormone. It has also been achieved that some plants, such as potatoes or strawberries, can better withstand frost, for example.

By BQUB14-Mtejado (Own work) [CC BY-SA 4.0], via Wikimedia Commons

Polymerase chain reaction (PCR)

Using this technique, it is possible to generate many copies of DNA from a DNA fragment. It was developed in 1983 by Kary Mullis in 1983, and allows you to clone DNA fragments without using any cells, directly in a test tube.

For this reaction it is necessary:

• The enzyme DNA polymerase, resistant to heat.
• A primer. A small RNA fragment of about 20 nucleotides, necessary for DNA polymerase to act.
• A source of heat.
• DNA nucleotides.

The DNA molecule to be copied is heated to a temperature higher than 90 ºC, so that it becomes denatured and loses its structure, separating the two chains that form the double helix. The hydrogen bonds that join the nitrogenous bases of both chains are broken.

Each of the two chains serves as a template for a new complementary chain.

The primer, an RNA fragment, allows the DNA polymerase enzyme to continue adding deoxyribonucleotides (DNA nucleotides) to form the entire complementary strand.

The newly formed chains are then separated again by the effect of heat, starting a new cycle.

Thus, in a short time many copies of the original DNA fragment can be obtained.