PCR Technique: Amplifying DNA for Genetic Analysis

Polymerase Chain Reaction (PCR)

Continuous replication is a fragment of DNA containing a gene or genes of our interest. In this way, we obtain a large number of copies that make it possible to study without worrying about the amount of the sample.

At first, it was a cumbersome technique. Due to the different temperatures used during the process, some of them very high, we were forced to replace the polymerase at each change in temperature. This problem was solved by the DNA polymerase of a thermophilic bacterium: Thermus aquaticus (Taq polymerase).

The method is deceptively simple and involves replication in a medium rich in triphosphate nucleotides, the DNA strand of interest, to which we add small pieces of RNA and Taq polymerase.

A cyclic temperature rise (the two DNA strands separate) and lowering (when fixing the primers and the polymerase can add triphosphate nucleotides) occurs. Each cycle doubles the number of copies of DNA, yielding a million copies in 20 cycles.

Currently, the reagent mixture is inserted into a device that will regulate the temperature (thermal cycler) and set the desired number of cycles (depending on the total amount of DNA we need, as shown) and the duration of each cycle (which depends directly on the length of the DNA strand to amplify).

This technique has proven indispensable in genetic engineering because it allows work from negligible amounts of DNA, with applications in gene cloning, evolutionary studies (fossil DNA), criminal investigation, paternity testing, etc.

PCR can also amplify highly repeated nucleotide sequences in our genome that have such a variety of combinations, which are authentic "genetic fingerprints." An individual can be identified precisely, or bonds of kinship between two individuals can be established. This makes them particularly interesting in police or forensic medicine.

Most enzymes are denatured at temperatures above 40ºC.

Raw material in the synthesis of DNA.

Recall that to begin to act, the polymerase has to add the first nucleotide on a previously formed RNA chain (primer).

Procurement of Vaccines

The traditional methods of vaccine production can be summarized as:

• Attenuating the virulence of the organism, usually with formaldehyde.
• Killing microorganisms, usually by heat.
• Inactivating the toxins produced by microorganisms while retaining their immunogenicity: toxoids.

Collection of Sera

Obtained from animals that were inoculated with the organism, and its antibodies are extracted.

Production of Antibiotics

That can be summarized as follows:

1. Cultivation of the mushroom producer in optimal conditions (Penicillium chrysogenum in the case of penicillin).
2. Purification of the antibiotic, usually by physical processes such as centrifugation or filtration.
3. Crystallization of the compound.