What makes pcr specific

In: Facts Methods and Technology. What is PCR? He was awarded the Nobel Prize in Chemistry in for his pioneering work. PCR is a common tool used in medical and biological research labs. It is used in the early stages of processing DNA for sequencing , for detecting the presence or absence of a gene to help identify pathogens during infection, and when generating forensic DNA profiles from tiny samples of DNA. How does PCR work?

We will explain exactly what each of these do as we go along. PCR involves a process of heating and cooling called thermal cycling which is carried out by machine.

There are three main stages: Denaturing — when the double-stranded template DNA is heated to separate it into two single strands. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.

The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis. This phenomenon leads to exon elimination at the time of excision of the introns and leads to the expression of different proteins from the same gene. It follows that depending on the cell type and regulatory profiles, we may not be dealing with the same transcript.

It is nevertheless very interesting to clone a transcript since its nucleotide sequence corresponds to the amino acid sequence resulting from the translation. On the other hand, with a cDNA, it is easier to carry out the expression of the gene and thus the functional evaluation of the corresponding protein or proteins in a cellular model of expression.

Very frequently, PCR cloning is practiced in parallel on genomic DNA genomic library and different cDNA libraries so as to determine the complete sequence of the gene, its expression profile, the modalities of splice regulation [ 8 , 39 ], etc.

This reaction is catalyzed by retrovirus reverse transcriptase reverse transcriptase which synthesizes a DNA chain from an RNA template. At first, the total RNAs are extracted. The following steps are carried out in the enclosure of the thermal cycler.

Other cycles are repeated to amplify double-stranded cDNAs in large quantities. In a given cell phenotype, an estimated 10—15, genes are expressed in humans and most mammals. Some cell transcripts are expressed at a few hundred or even a few thousand copies per cell, but the majority of transcripts represent a low copy number. The expression profiles of transcripts undergo qualitative or quantitative variations that reflect the biological dynamics of the cell. The identification of variations in gene expression in a given physiological or pathological context can therefore provide valuable information concerning the function of genes and the influence of modulation factors on their expression, whether they are physiological or of environmental origin.

The analysis of the expression variations of genes involved in a pathology can lead to new therapeutic or diagnostic targets. Finally, from a fundamental point of view, studying the gene expression profile makes it possible to advance in understanding the mechanisms of cellular physiology [ 40 , 41 , 42 ]. The method is based on the detection of a fluorescent signal that is produced in proportion to the amplification of the PCR product, cycle after cycle.

It requires a thermal cycler coupled to an optical reading system that measures fluorescence emission. A nucleotide probe is synthesized so that it can hybridize selectively to the DNA of interest between the sequences where the primers hybridize. As long as the two fluorochromes remain present at the probe, the extinguisher prevents the fluorescence of the signal. In this step, the proximity of the quencher and the signal induces a lack of fluorescence emission.

The level of fluorescence then released is proportional to the amount of PCR products generated in each cycle. The thermal cycler is designed so that each sample the PCR is generally carried out in well plates is connected to an optical system.

This includes a laser transmitter connected to an optical fiber. The laser, via the optical fiber, excites the fluorochrome within the PCR reaction mixture. The fluorescence emitted is retransmitted, always through optical fiber, to a digital camera connected to a computer.

A software then analyzes and stores the data. Quantitative PCR is a method of high specificity and sensitivity. It is very timely for countless applications. It is indeed very often used for this purpose, for example, in order to determine the viral load, in particular in cases of hepatitis C or AIDS.

One of the most remarkable and useful applications is the analysis of gene expression through the quantitative measurement of transcripts. These standards can be internal or external. External standards may be homologous or heterologous. The standard is an RNA more rarely a DNA which is present in the RNA extract internal standard or which is added in known quantity in the reaction mixture external standard.

The standard is amplified at the same time as the RNA of interest. There is therefore competition between the amplification of the standard and that of the DNA of interest. The higher the standard quantity, the less the RNA of interest will be amplified and therefore its quantity will be small. Of course, the method of analysis of the PCR sample must make it possible to discriminate the standard with respect to the RNA of interest on the one hand and on the other hand to evaluate the relative amount of DNA of interest by comparison with the amount of standard that is known [ 48 ].

The internal standards are endogenous RNA, corresponding to RNA genes whose expression is presumed constant actin, beta2-microglobulin, etc. These standards have a major disadvantage: they require the use of primers different from those used for the RNA of interest. The kinetics of amplification are therefore substantially different, and it is very difficult or impossible to guarantee a constant expression between different samples.

The homologous external RNA standards are synthetic RNAs that share the same priming hybridization sites as the RNA of interest and that have the same overall sequence, with a slight mutation, deletion, or insertion that will allow the identification and quantification thereof with respect to the signal rendered by the RNA of interest.

These standards make it possible on the one hand to appreciate the variability introduced at the level of the RT and, on the other hand, generally have the same amplification efficiency as the RNA of interest whether it is at the RT level or PCR [ 48 , 49 ].

However, unlike homologous external standards, they have a different amplification efficiency compared to that of the RNA of interest. A dilution series is performed, each being used for amplification. It is then a question of defining the ideal number of cycles to be placed in the exponential phase of the reaction while ensuring an effective amplification.

Knowing the value of the signal measured on the sample to be quantified, the corresponding number of copies can be extrapolated from the curve. In the case of competitive PCR, a series of synthetic external homologous standard RNA dilutions are co-amplified with equivalent amounts of total RNA and thus an equivalent amount of the native gene [ 50 , 51 ].

The standard competes with the RNA of interest for polymerase and primers. As the standard concentration increases, the signal of the gene of interest decreases. Here, the PCR does not need to be performed in the exponential phase and the results show a correct reproducibility. However, the method is cumbersome and does not allow to manage many samples simultaneously [ 52 ]. PCR is a fabulous diagnostic tool. It is already widely used in the detection of genetic diseases.

The amplification of all or part of a gene responsible for a genetic disease makes it possible to reveal the deleterious mutations s , their positions, their sizes, and their natures.

It is thus possible to detect deletions, inversions, insertions, and even point mutations, either by direct analysis of PCR products by electrophoresis or by combining PCR with other techniques [ 53 ].

But PCR can still be used to detect infectious diseases viral, bacterial, parasitic, etc. Although other diagnostic tools are effective at detecting these diseases, PCR has the enormous advantage of producing very reliable and rapid results from minute biological samples in which the presence of the pathogen is not always detectable with other techniques [ 53 , 54 ]. In the context of genetic diseases, it is a question of detecting a mutation on the sequence of a gene.

Several situations arise. The simplest ones concern insertions and deletions. In these cases, the mutation is manifested by the change in the size of the gene or part of the gene.

Insofar as the mutation is known and described, it suffices to amplify all or part of the gene. A deletion presents a contrary result [ 55 ]. The analysis of PCR products by electrophoresis, and therefore the evaluation of their size, leads directly to the diagnosis.

The detection of inversions and point mutations is more delicate. The difference in size between healthy and diseased DNA is zero in the case of an inversion and almost zero in the case of a point mutation. We cannot therefore retain the size criterion of the PCR products to achieve the result.

It is therefore necessary to resort to techniques complementary to PCR. Three approaches can be selected, the southern blot, the restriction fragment length polymorphism RFLP , or the detection of mismatch. The southern blot consists in hybridizing on the PCR product an oligonucleotide probe marked, thanks to a radioactive isotope or a fluorochrome, whose sequence is complementary and therefore specific to that which corresponds to the mutation.

This strategy is well suited to inversion cases [ 56 , 57 ]. The RFLP can detect inversions such as point mutations. One modification of conventional PCR allows researchers to copy a particular DNA sequence and quantify it simultaneously.

This refinement involves the use of fluorescent dyes or probes that label double-stranded DNA molecules. These fluorescent markers bind to the new DNA copies as they accumulate, making "real-time" monitoring of DNA production possible.

As the number of gene copies increases with each PCR cycle, the fluorescent signal becomes more intense. Plotting fluorescence against cycle number and comparing the results to a standard curve produced by real-time PCR of known amounts of DNA enables scientists to determine the amount of DNA present during each step of the PCR reaction.

This page appears in the following eBook. Aa Aa Aa. What is PCR? PCR makes it possible to produce millions of copies of a DNA sequence in a test tube in just a few hours, even with a very small initial amount of DNA.

Since its introduction, PCR has revolutionized molecular biology, and it has become an essential tool for biologists, physicians, and anyone else who works with DNA. How does PCR work? Step 1: Denaturation. Figure 2: When heated, the DNA strands separate. Step 2: Annealing. Figure 3: When the solution is cooled, the primers anneal.