**3. The reaction conditions**

The volumes of reaction medium vary between 10 and 100 μl. There are a multitude of reaction medium formulas. However, it is possible to define a standard formula that is suitable for most polymerization reactions. This formula has been chosen by most manufacturers and suppliers, who, moreover, deliver a ready-to-use buffer solution with Taq polymerase. Concentrated 10 times, its formula is approximately the following: 100 mM Tris-HCl, pH 9.0; 15 mM MgCl2, 500 mM KCl [2, 4].

It is possible to add detergents (Tween 20, Triton X-100) or glycerol in order to increase the conditions of stringency that make it harder and therefore more selective hybridization of the primers. This approach is generally used to reduce the level of nonspecific amplifications due to the hybridization of the primers on sequences without relationship with the sequence of interest. We can also reduce the concentration of KCl until eliminated or increase the concentration of MgCl2 [1, 5]. Indeed, some pairs of primers work better with solutions enriched with magnesium. On the other hand, with high concentrations of dNTP, the concentration of magnesium should be increased because of stoichiometric interactions between magnesium and dNTPs that reduce the amount of free magnesium in the reaction medium. dNTPs (deoxyribonucleoside triphosphates) provide both the energy and the nucleotides needed for DNA synthesis during the chain polymerization. They are incorporated in the reaction medium in excess, that is, about 200 μM final. Depending on the reaction volume chosen, the primer concentration may vary between 10 and 50 pmol per sample. Matrix DNA can come from any organism and even complex biological materials that include DNAs from different organisms. But to ensure the success of a PCR, it is still necessary that the DNA matrix is not too degraded. This criterion is obviously all the more crucial as the size of the sequence of interest is large. It is also important that the DNA extract is not contaminated with inhibitors of the polymerase chain reaction (detergents, EDTA, phenol, proteins, etc.) [6, 7]. The amount of template DNA in the reaction medium initiate that the amplification reaction can be reduced to a single copy. The maximum quantity may in no case exceed 2 μg. In general, the amounts used are in the range of 10–500 ng of template DNA. The amount of Taq polymerase per sample is generally between 1 and 3 units. The choice of the duration of the temperature cycles and the number of cycles depends on the size of the sequence of interest as well as the size and the complementarity of the primers. The durations should be reduced to a minimum not only to save time but also to prevent risk of nonspecific amplification. For denaturation and hybridization of primers, 30 seconds are usually sufficient. For elongation, it takes 1 minute per kilobase of DNA of interest and 2 minutes per kilobase for the final cycle of elongation. The number of cycles, generally between 20 and 40, is inversely proportional to the abundance of DNA matrix [6–8].

#### **4. PCR product detection and analysis**

The product of a PCR consists of one or more DNA fragments (the sequence or sequences of interest). The detection and analysis of the products can be very quickly carried out by agarose gel electrophoresis (or acrylamide). The DNA is revealed by ethidium bromide staining [2, 3, 5]. Thus, the products are instantly

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attention.

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

visible by ultraviolet transillumination (280–320 nm). Very small products are often visible very close to the migration front in the form of more or less diffuse bands. They correspond to primer dimers and sometimes to the primers themselves. Depending on the reaction conditions, nonspecific DNA fragments may be amplified to a greater or lesser extent, forming net bands or "smear" [6–9]. On automated systems, a fragment analyzer is now used. This apparatus uses the principle of capillary electrophoresis. Fragment detection is performed by a laser diode. This is only possible if the PCR is performed with primers coupled to

**5. Overview of molecular techniques based on PCR technology**

(VNTR) polymorphisms. Amplified fragment length polymorphisms.

Microsatellites are hypervariable; on a locus, they often show dozens of different alleles from each other in the number of repetitions. They are still the markers of choice for studies on the diversity, paternity analysis and mapping of quantitative effects loci (QTL), although this could change, in the near future, through the elaboration inexpensive SNP assay methods. Minisatellites have the same characteristics as microsatellites, but repetitions range from ten to a few hundred base pairs. Micro- and minisatellites are also known as variable number of tandem repeat

Microsatellites are now the most used markers in genetic characterization studies of farmed animals [11]. The high mutation rate and codominant nature favor the estimation of intra and interracial diversity, and the genetic mixing between races, even if they are very close. Challenges have surrounded the choice of a mutation model—the infinite or progressive allele mutation model [12] for the analysis of microsatellite data. However, simulation studies have indicated that the infinite allele mutation model is generally valid for the evaluation of intraracial diversity [13]. The low number of alleles per population and observed and expected heterozygosity are the most commonly used parameters for assessing intraracial diversity. The simplest parameters for evaluating interracial diversity are genetic differentiation or fixation indices. Several estimators have been proposed (e.g., FST—fixation index and GST—glutathione S transferase), and the most widely used is FST [14], which measures the degree of genetic differentiation of subpopulations by calculation standardized variances of allele frequencies of populations. Statistical significance is calculated for FST values between population pairs to test the null hypothesis of a lack of genetic differentiation between populations and, consequently, the partitioning of genetic diversity [15]. Microsatellite data are also commonly used to assess genetic relationships between populations and subjects through the estimation of genetic distances [16–19]. The measure of genetic distance used most often is the standard genetic distance of Nei [20]. In another case, the modified Cavalli-Sforza distance is recommended [21] for the closest populations, where genetic drift is the main factor of genetic differentiation. The genetic relationship between breeds is often visualized by the reconstruction of a phylogeny, most often using the "neighbor-joining" method [22]. However, the main problem in the reconstruction of the phylogenetic tree is that the evolution of the lines is presumed to be uncrosslinked that is to say that the lines can deviate, but can never come from interbreeding. This assumption is rarely valid for farm animals, as new breeds are often derived from crosses between two or more ancestral breeds. The visualization of breed evolution by phylogenetic reconstruction must therefore be interpreted with great

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

fluorochromes [10].

**5.1 Microsatellites**

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

visible by ultraviolet transillumination (280–320 nm). Very small products are often visible very close to the migration front in the form of more or less diffuse bands. They correspond to primer dimers and sometimes to the primers themselves. Depending on the reaction conditions, nonspecific DNA fragments may be amplified to a greater or lesser extent, forming net bands or "smear" [6–9]. On automated systems, a fragment analyzer is now used. This apparatus uses the principle of capillary electrophoresis. Fragment detection is performed by a laser diode. This is only possible if the PCR is performed with primers coupled to fluorochromes [10].
