Techniques

Polymerase chain reaction, better known as PCR, is a multiplicative technique ("amplifying" is actually the term that has been imposed) small amounts of DNA between hundreds of thousands and millions of times. Because of its high sensitivity, this technique allows a gene to be identified from a single hair, a somatic cell or a spermatozoon.

PCR is basically a DNA amplifying technique.

This method is based, in its simplest form, in the performance of three successive reactions carried out at different temperatures.

• Denaturalization

94-95°C por 45 segundos si G+C < 55%.

• Hybridization temperature (Annealing)

It must be empirically calculated or determined for each pair of primers.

Too high = little or no product at all.

• Extension

At the optimum temperature of the used DNA polymerase.

Eg.: 72°C for Taq -------> 1 min/kb of length, since the Taq polymerizes 2000 pb/min

Note: Only from the third cycle DNA duplex are produced with the desired length, which from there, they become the main product.

Since RNA is usually single-strand and sensitive to heat, it is necessary to make a reverse transcription (RT) before starting the PCR amplification. The reverse transcription generates a copy of the RNA strand, but this copy is complementary DNA (cDNA), which is stable to heat and can resist the PCR methodology.

It is frequently used to detect infectious agents e.g.: RNA virus.

It can be used to build cDNA libraries.

The RT-PCR steps are:

• Binding of the primer to the target RNA sequence.
• Reverse Transcription: The rTth polymerase catalyzes the extension of the primer by incorporating complementary nucleotides.
• End of reverse transcription, the strand of the cDNA complementary to the RNA is obtained.
• </>PCR is performed.

RFLP (Restriction Fragment Length Polymorphism). This technique, developed in the late 1970s, is based on the detection of DNA fragments of different molecular weight or different sizes (by restriction enzyme digestion). It is used to differentiate or genotype microorganisms by analyzing patterns derived from DNA cuts.

When PCR is used to amplify DNA fragments and after restriction enzymes, it is known as PCR-RFLP. And it is widely used to genotyping viruses such as Human Papilloma Virus, Hepatitis C Virus among others.

• GENOTYPING BY RFLP described by Davidson and cols., J,G.Virol, 76, 1995. 

With this methodology it is possible to detect the presence of two or more microorganisms simultaneously in a single reaction. This modification allows to quickly discard the infection by the pathogens usually responsible for the pictures to be diagnosed. In order to take advantage of all the benefits of this technique, it is necessary to carefully respect the sampling instructions, conservation and conditions for the shipment to the laboratory, as it is a very sensitive reaction to the presence of inhibitors and contaminants.

• Describes a PCR in which there is multiple primers pairs which results in a series of products. They can be seen as multiple bands on an agarose gel.
• Multiplex PCR is frequently used in a medical diagnosis.
• It saves sample, time and expense.
• It saves sample, time and expense.

PCR of SYNCYTIAL RESPIRATORY VIRUS A and B, INFLUENZA A, B and C</>

This modification is about an amplification of a portion of a product from a previously performed PCR. The obtained product is comprised within the sequence of the product of the first round. This modification allows to significantly increase the sensitivity and specificity of the PCR reaction.

• Sometimes 1 round of PCR does not give a unique product because we start from a temperate complex, or we want to improve sensitivity. It can be solved using a second pair of primers that internally hybridize a little more than the first ones, performing a second round of PCR using the product of the first round.
• It yields a unique product because only the correct DNA fragment possesses the hybridization sites for the second pair of primers.

The Real Time PCR combines the new automatic systems for the nucleic acids purification, it offers an ideal platform for the development of a wide variety of molecular tests for the identification and quantification of infectious agents of clinical interest.

This technology is based on the detection of the emission of fluorescence during each amplification cycle, which allows the continuous monitoring of the PCR reaction. An exciter laser beam is transmitted to the sample by an optical fiber.

In the Real Time PCR, the amplification and detection processes occur simultaneously in the same closed vial, without the need for any further action. By fluorescence detection the amount of DNA formed can be measured during the amplification.

There can be two types of fluorescence detection systems, used in real-time PCR:

1. Intercalating agents.

2. Specific probes labeled with fluorochromes.

They are fluorochromes that significantly increase the emission of fluorescence when they bind to double-stranded DNA. The increase of DNA in each cycle is reflected in a proportional increase of the emitted fluorescence. This detection system has the advantage that the optimization of the reaction conditions is very easy. Their drawback is their low specificity, because they bind differently to nonspecifically generated products. To improve this, optimum reaction conditions and primer selection must be employed to decrease the risk of dimer formation.

They are probes labeled with two types of fluorochromes, one donor and one acceptor. The process is based on the transfer of fluorescent energy by resonance between the two molecules.

FRET probes are composed of two probes that bind to adjacent sequences of the target DNA. One of the probes carries a donor at the 3' end and the other an acceptor at the 5' end. When the probes are hybridized, both fluorochromes are close. Upon being excited, the donor transfers its energy to the acceptor, which, at the same time, emits the fluorescence that detects the reader of the equipment.

The use of probes guarantees the specificity of detection and allows the identification of polymorphisms or specific mutations.

Real-time PCR offers an ideal platform for the development of a wide variety of molecular tests, due to its undoubted advantages, such as ease of use, thanks to its speed; it allows a much larger flow of samples and tests. Another advantage is that, when using closed systems, the risk of contamination significantly decreases. In addition, it allows quantifying the initial concentration of nucleic acid present in the samples more accurately and simply.