How many primers are used in a pcr reaction

Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: 1 they can generate new strands of DNA using a DNA template and primers, and 2 they are heat resistant. Primers - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer. Only during the exponential phase of the PCR reaction is it possible to extrapolate back to determine the starting quantity of the target sequence contained in the sample.

Because of inhibitors of the polymerase reaction found in the sample, reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify target sequence at an exponential rate and a "plateau effect" occurs, making the end point quantification of PCR products unreliable.

A complete PCR reaction can be performed in a few hours, or even less than an hour with certain high-speed machines. After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.

Related Content:. What is DNA replication? What is DNA? What is a gene? What is DNA sequencing? How helpful was this page? What's the main reason for your rating? Which of these best describes your occupation? What is the first part of your school's postcode? How has the site influenced you or others? 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.

It involves a restriction enzyme capable of hydrolyzing the PCR product at the sequence which sets the mutation. This approach is only possible if a restriction site is indeed present on this sequence, whether it is the mutated allele or the wild-type allele.

Mismatch detection is, like the RFLP, adapted to inversions and point mutations [ 57 , 58 , 59 ]. This mixture is then denatured by the temperature and then rehybridized. The mismatches concern a single base pair in the case of a point mutation and several base pairs in the case of an inversion.

These mismatches are then degraded by S1 nuclease, an enzyme that degrades only single-stranded DNAs. Another solution is to cleave the mismatches chemically osmium tetroxide, then piperidine , but it is more suitable for point mutations. In summary, mutation induces a mismatch at the level of enzymatic or chemical cleavage which leads to the generation of two fragments from a single PCR product.

These fragments are analyzed by electrophoresis. Contamination with viruses or microorganisms bacteria, parasites, etc. PCR is therefore a tool all the more effective in detecting the presence of a pathogen in a biological sample that its sensitivity and specificity are very large.

The performance of the PCR diagnosis is essentially based on a criterion: the choice of primers capable of very selectively amplifying a sequence of the DNA of the virus or microorganism [ 57 , 58 , 59 ]. Matrix DNA, on the other hand, must be extracted from a tissue in which the microorganism is present.

It is therefore sufficient to amplify a specific sequence of the pathogen from a sample taken on the patient and to analyze the PCR product by electrophoresis. The size of the amplified DNA fragment, which must conform to the expected size, guarantees the reliability of the result and therefore of the diagnosis.

This method, quite reliable and inexpensive, nevertheless has some disadvantages. False positives are quite common because of cross-reactivities.

Positive samples are therefore tested for control by another routine technique, Western blot. The blood of these newborns usually contains anti-HIV antibodies of maternal origin and they are therefore seropositive.

On the other hand, they do not necessarily carry the virus. In this type of case, the PCR diagnosis is relevant [ 57 , 58 , 59 , 60 ]. The method involves amplifying a specific sequence of the provirus from a lymphocyte extract. The same principle is used for the detection of toxoplasma in newborns whose mother is a carrier.

Quantitative or semi-quantitative methods have been developed which also make it possible to evaluate the viral load. PCR is remarkably effective at identifying species, varieties, or individuals by genetic fingerprinting.

This application is based on the knowledge acquired on genome structure. It is simply to amplify nucleotide sequences that are specific to species, variety, or individual.

In eukaryotes, in particular, these sequences are very numerous and offer a vast palette that allows identification in a very precise and very selective way. Indeed, the genomes of eukaryotic organisms have, unlike prokaryotes, coding sequences and noncoding sequences. The coding sequences correspond to the genes and are therefore translated into proteins. The coding sequences are highly homologous in individuals of the same species. Indeed, the species is characterized by characters and common traits that are guaranteed by its genes.

The phenotypic differences between the individuals that compose it are based on the allelic variations and the different alleles of the same gene show sequence differences that are minute of the order of 1 base pair per [ 61 , 62 ]. From one species to another, depending on the phylogenetic distance that separates them, the sequences of the genes that code for the same function have very strong homologies, all the more so that the function of the gene is essential to the embryogenesis or metabolism.

As a result, coding sequences are of little relevance in terms of identification. On the other hand, the noncoding sequences are very polymorphous between species as between individuals of the same species. They thus present a large choice of genetic markers that make it possible to establish identification tests which are highly discriminating.

Among these markers are minisatellites or variable number of tandem repeats and microsatellites or STR, short tandem repeats [ 61 , 62 , 63 ]. The amplification products are then either analyzed by electrophoresis or undergo fragment analysis using a capillary sequencer.

The variety of amplification products obtained leads to footprints that are specific individuals. DNA fingerprinting has become much more commonplace in recent years in the context of judicial investigations. But these techniques are equally as effective in other species as humans and allow not only identifying individuals but also varieties or species. The type of identification depends simply on the choice of markers. Similarly, for varietal identification purposes, one can commonly proceed according to protocols derived from the PCR [ 64 , 65 , 66 ].

These primers will hybridize randomly, but PCR usually results in an electrophoresis amplification profile which is specific to the variety from which the matrix DNA is derived. Amplification of fragment length polymorphism AFLP is a much more efficient method. It first consists hydrolyzing the genomic DNA with one or better two restriction endonucleases.

Then, we proceed with the ligation of adapters defined sequences of DNA of about 15 nucleotides at the level of the generated cohesive ends by restriction enzymes.

Finally, the product of the ligation is amplified by PCR with a pair of primers that hybridizes at the level of the adapters. However, the AFLP shows cleaner and more reproducible results. This is the most successful method to date applied to varietal identification.

The extension of genotyping approaches to all living organisms has made significant advances in the reconstruction of the history of life. At the population level, the distribution and frequency of known genetic polymorphisms in a species can highlight the evolving forces at play, reveal the effects of natural selection, and infer demographic change. Moreover, the comparison of the sequences of the same genes between different species and that of whole genomes is at the origin of the molecular phylogenies that currently prevail in the classification.

They make it possible to trace the relationships between species on the basis of the divergence of their DNA sequences. As such, the PCR is a key stage at two levels. The first concerns the isolation of homologous genes in several species and their characterization. The second is the production of amplified total genomic DNA for genome sequencing and comparative analysis.

But PCR is also used to identify the genetic heritage of missing organisms. The DNA breaks down by fragmentation after the death of the body.

If we can recover these fragments and amplify them, it becomes possible, in spite of its state, to deduce all or part of the initial genome of the individual.

PCR has thus become the primary tool in the field of palaeogenetics, which consists in recovering and analyzing DNA sequences of more or less old organisms, and this as well from the remains preserved in museum collections, from historical site where the skeletal or mummified remains of extinct organisms for hundreds thousands or even hundreds of thousands of years. The uses of the PCR thus quickly stopped being limited to the studies of biology, to gain other disciplines or fields of activities.

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