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

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 (VNTR) polymorphisms. Amplified fragment length polymorphisms.

#### **5.1 Microsatellites**

*Synthetic Biology - New Interdisciplinary Science*

polymerase [4].

**3. The reaction conditions**

which are usually sufficient to denature most proteins. *Thermus aquaticus* finds its temperature of comfort at 72°C, optimum temperature for the activity of its

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].

**150**

**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

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 attention.

### **5.2 SNP**

Single nucleotide polymorphisms (SNPs) are used as an alternative to microsatellites in genetic diversity studies. Several technologies are available to detect the type of SNP markers [23]. As biallelic markers, SNPs have relatively low amounts of information, and to reach the information level of a standard panel of 30 microsatellite loci, larger amounts must be used. However, ever-evolving molecular technologies increase automation and reduce the cost of typing SNPs, which will likely allow, in the near future, the parallel analysis of a large number of markers at a reduced cost. In this perspective, large-scale projects are being implemented for several livestock species to identify millions of SNPs [24] and validate several thousands and identify haplotype in the genome. As with sequence information, SNPs allow for direct comparison and joint analysis of different experiments. SNPs are likely to be interesting markers for future use in genetic diversity studies because they can be easily used in the assessment of functional or neutral variation. However, the preliminary phase of SNP discovery or selection of SNPs from databases is critical. SNPs can be generated through various experimental protocols, such as sequencing, single-stranded coformational polymorphism (SSCP) or denaturing high-performance liquid chromatography (DHPLC) or in silico, aligning and comparing multiple sequences from the same region from public databases on genomes and sequential expression tags (ESTs). If the data were obtained randomly, the standard population genetic parameter estimators cannot be applied. A common example is when SNPs initially identified in a small sample (panel) of individuals are then typed into a larger sample of chromosomes. By preferably performing sampling of SNPs at intermediate frequencies, such a protocol will affect the distribution of allele frequencies with respect to the probable values for a random sample. SNPs present a modern tool in the context of genetic analyzes of the population; however, it is necessary to develop statistical methods that will take into account each SNP operating method and their locations [25, 26].

### **5.3 Amplification of fragment length polymorphism (AFLP)**

AFLPs are dominant biallelic markers [27]. Variations on many loci can be arranged simultaneously to detect single nucleotide variations of unknown genomic regions, where a given mutation may often be present in undetermined functional genes. The disadvantage is that they show a dominant mode of inheritance, which reduces their power during genetic analyses of the population on intraracial diversity and consanguinity. However, AFLP profiles are highly informative in the evaluation of race relations [28–32] and related species [33].

#### **5.4 Restriction fragment length polymorphism (RFLP)**

Restriction fragment length polymorphisms (RFLPs) are identified using restriction enzymes that cut DNA only at specific "restriction sites" (e.g., EcoRI cuts at the site defined by the palindrome GAATTC sequence). At present, the most common use of RFLPs is downstream PCR (PCR-RFLP) to detect alleles that differ in sequence at a given restriction site. A gene fragment is first amplified using PCR and then exposed to a specific restriction enzyme that cuts only one of the allelic forms. The digested amplicons are usually resolved by electrophoresis. Microsatellites or SSRs (simple sequence repeats) or STRs (short tandem repeats) consist of a few nucleotides—2–6 base pair DNA sequence—epeated several times in tandem (e.g., CACACACACACACACA). They are spread on a eukaryotic genome. Microsatellites are relatively small in size and, therefore, are easily

**153**

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

amplified using DNA PCRs extracted from different sources, such as blood, hair, skin, or even feces. Polymorphisms can be visualized on a sequencing gel, and the availability of automated DNA sequencers allows high-throughput analysis of a

Mitochondrial DNA polymorphisms (mtDNA) have been widely used in analyzes of phylogenetic and genetic diversity. The haploid mtDNA transported by the mitochondria of the cellular cytoplasm has a maternal mode of inheritance (the animals inherit the mtDNA from their mothers and not from their fathers) and a high mutation rate; it does not recombine. These features allow biologists to reconstruct intra and interracial evolutionary relationships by evaluating mtDNA mutation patterns. mtDNA tags can also provide a quick way to detect hybridization between farmed species and subspecies [36]. Polymorphisms in the hypervariable region of the D-loop or the mtDNA control region have largely contributed to the identification of wild ascendants of domestic species and to the establishment of

This is one of the most remarkable applications of PCR. It makes it possible to isolate, that is to say, to purify a gene without resorting to traditional methods of molecular cloning which consist in inserting a DNA library in a plasmid vector which is then used to transform a bacterial strain whose clones after selection are screened. The realization is much faster and much less random using PCR. Acellular

cloning is used when using PCR because it is useless to use a cellular system (bacteria, yeast, and animal or plant cell) to amplify the clone. The realization of molecular cloning by PCR depends on two major criteria: the choice of DNA extract (matrix DNA) and primers. It is indeed essential to have more or less reliable data on the sequence of the gene that is to be cloned and/or flanking sequences in order to synthesize the sets of primers necessary for its amplification in whole or in part. On the other hand, is it still necessary to perform the PCR on the appropriate matrix DNA [37, 38]. We can choose the genomic DNA that includes the total sequence of the genome and therefore all the genes of the species. In this case, the genes include both exons and introns and their amplification results in the cloning of the complete gene sequence and even, depending on the primers that have been chosen, regulatory regions. But we can also choose to extract the messenger RNA (mRNA), that is to say the only coding sequences of the gene—the transcripts. Since RNAs are unstable, messenger RNAs are transformed into complementary DNA (cDNA) by RT-PCR (see below), a variant of PCR that uses reverse transcriptase and allows changing the RNA sequences into DNA. It is on this cDNA library that PCR is then performed to clone the gene of interest. In this case, the deal is more complex. The presence of the gene transcript in the extract depends on the cell type, tissue, or organ from which the mRNA extraction was performed. Indeed, transcription is specific to the cell type. More serious, the expression of a gene is often regulated by physiological factors, environmental, in this case the gene of interest is not necessarily transcribed and the cDNA library may not contain it. Finally, it must be said that transcription is itself regulated and is often accompanied by alternative splicing. This phenomenon leads to exon elimination at the time of excision of the

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

large number of samples [34, 35].

**5.5 Mitochondrial DNA markers**

geographical models of genetic diversity.

**6. Applications**

**6.1 Acellular cloning**

amplified using DNA PCRs extracted from different sources, such as blood, hair, skin, or even feces. Polymorphisms can be visualized on a sequencing gel, and the availability of automated DNA sequencers allows high-throughput analysis of a large number of samples [34, 35].
