**6.5 PCR applied to diagnosis**

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.), as is already the case for AIDS, hepatitis C, or chlamydia infections. 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].

## **6.6 Detection of genetic diseases**

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. In the case of an insertion, the PCR product from a patient's DNA is longer than that from a healthy person. 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].

**157**

*Polymerase Chain Reaction (PCR): Principle and Applications*

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. The restriction enzyme thus hydrolyzes either the PCR product derived from healthy DNA or that which is derived from the diseased DNA. From these PCR products, one or two DNA fragments are thus obtained which are then revealed by electrophoresis. Mismatch detection is, like the RFLP, adapted to inversions and point mutations [57–59]. The PCR product from the patient's DNA (sample DNA) is mixed with the PCR product from the DNA of a healthy person (reference DNA). This mixture is then denatured by the temperature and then rehybridized. Yes the sample DNA is mutated; the pairings between sample DNA and reference DNA will be incomplete at the level of the mutation. 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.) necessarily results in the presence of their genetic material in all or part of the infected organism. 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 [57–59]. 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. In the case of AIDS (HIV) testing, for example, routine testing is based on the ELISA method of detecting HIV antibodies or viral antigens in the patient's serum by an immunoassay technique. 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. There remains the problem of HIV-positive people who do not carry the virus, such as children whose mothers have AIDS. 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 [57–60]. 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. It is of course possible to diagnose AIDS by RT-PCR by looking for viral RNA in the patient's serum. 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

*DOI: http://dx.doi.org/10.5772/intechopen.86491*

**6.7 Detection of infectious diseases**

**6.8 PCR applied to identification**

*Polymerase Chain Reaction (PCR): Principle and Applications DOI: http://dx.doi.org/10.5772/intechopen.86491*

*Synthetic Biology - New Interdisciplinary Science*

**6.5 PCR applied to diagnosis**

**6.6 Detection of genetic diseases**

This strategy is well suited to inversion cases [56, 57].

have a different amplification efficiency compared to that of the RNA of interest. In the case of quantitative RT-PCR (semi-quantitative PCR), the standard consists of a titrated solution of DNA of sequence identical to that of the DNA of interest to be quantified. 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. Then, each standard DNA dilution as well as the DNA extracted from the sample to be quantified are submitted in parallel to the PCR reaction. A standard curve is established with standard dilutions [signal = f (concentration)]. 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.), as is already the case for AIDS, hepatitis C, or chlamydia infections. 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. In the case of an insertion, the PCR product from a patient's DNA is longer than that from a healthy person. 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.

**156**

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. The restriction enzyme thus hydrolyzes either the PCR product derived from healthy DNA or that which is derived from the diseased DNA. From these PCR products, one or two DNA fragments are thus obtained which are then revealed by electrophoresis. Mismatch detection is, like the RFLP, adapted to inversions and point mutations [57–59]. The PCR product from the patient's DNA (sample DNA) is mixed with the PCR product from the DNA of a healthy person (reference DNA). This mixture is then denatured by the temperature and then rehybridized. Yes the sample DNA is mutated; the pairings between sample DNA and reference DNA will be incomplete at the level of the mutation. 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.

#### **6.7 Detection of infectious diseases**

Contamination with viruses or microorganisms (bacteria, parasites, etc.) necessarily results in the presence of their genetic material in all or part of the infected organism. 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 [57–59]. 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. In the case of AIDS (HIV) testing, for example, routine testing is based on the ELISA method of detecting HIV antibodies or viral antigens in the patient's serum by an immunoassay technique. 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. There remains the problem of HIV-positive people who do not carry the virus, such as children whose mothers have AIDS. 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 [57–60]. 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. It is of course possible to diagnose AIDS by RT-PCR by looking for viral RNA in the patient's serum. Quantitative or semi-quantitative methods have been developed which also make it possible to evaluate the viral load.

#### **6.8 PCR applied to identification**

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 noncoding sequences, which are therefore not translated, represent a large proportion of eukaryotic genomic DNA (up to 98%). 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 1000) [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) [61–63]. VNTRs and STRs are repetitive polymorphisms composed of sequences that are repeated in tandem. These repeat sequences measure from 10 to 40 base pairs for VNTRs and from 1 to 5 base pairs for STRs. From one individual to another, the repeated sequence of a VNTR or STR is identical but the number of repetitions and therefore the size of the VNTR or the STR can be very variable (we speak of alleles). On the other hand, there is a wide variety of VNTRs and STRs on eukaryotic genomes. Detection of STR or VNTR polymorphism is by PCR using primers that hybridize to nonpolymorphic flanking sequences. The amplification products are then either analyzed by electrophoresis or undergo fragment analysis using a capillary sequencer. It is now possible to simultaneously amplify several STRs or VNTRs by using several pairs of primers. The variety of amplification products obtained leads to footprints that are specific individuals. On the other hand, the power of PCR makes it possible to amplify micro- and minisatellites from very little DNA. 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 [64–66].

Two techniques that are relevant are the random amplification of polymorphic DNA (RAPD) and the amplification of fragment length polymorphism (AFLP). (Random amplification of polymorphic DNA (RAPD) is a PCR for varietal identification that uses pairs of random primers of reduced size (about 10 base pairs). 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. The

**159**

**Author details**

Genetic Resources, Degache, Tunisia

Applied on Agriculture, Ariana, Tunisia

provided the original work is properly cited.

\*Address all correspondence to: kadrikarim2001@yahoo.fr

1 Regional Research Center on Oasis Agriculture, Laboratory of Biotechnology and

2 National Institute for Agronomic Research in Tunis, Laboratory of Biotechnology

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Karim Kadri1,2

*Polymerase Chain Reaction (PCR): Principle and Applications*

AFLP gives a result comparable to the RAPD. However, the AFLP shows cleaner and more reproducible results. This is the most successful method to date applied to

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.

*DOI: http://dx.doi.org/10.5772/intechopen.86491*

varietal identification.

**7. Conclusion**

AFLP gives a result comparable to the RAPD. However, the AFLP shows cleaner and more reproducible results. This is the most successful method to date applied to varietal identification.
