**2. Principle of the PCR**

PCR makes it possible to obtain, by in vitro replication, multiple copies of a DNA fragment from an extract. Matrix DNA can be genomic DNA as well as complementary DNA obtained by RT-PCR from a messenger RNA extract (poly-A RNA), or even mitochondrial DNA. It is a technique for obtaining large amounts of a specific DNA sequence from a DNA sample. This amplification is based on the replication of a double-stranded DNA template. It is broken down into three phases: a denaturation phase, a hybridization phase with primers, and an elongation phase. The products of each synthesis step serve as a template for the following steps, thus exponential amplification is achieved [1].

The polymerase chain reaction is carried out in a reaction mixture which comprises the DNA extract (template DNA), Taq polymerase, the primers, and the four deoxyribonucleoside triphosphates (dNTPs) in excess in a buffer solution. The tubes containing the mixture reaction are subjected to repetitive temperature cycles several tens of times in the heating block of a thermal cycler (apparatus which has an enclosure where the sample tubes are deposited and in which the temperature can vary, very quickly and precisely, from 0 to 100°C by Peltier effect) [1, 2]. The apparatus allows the programming of the duration and the succession of the cycles of temperature steps. Each cycle includes three periods of a few tens of seconds. The process of the PCR is subdivided into three stages as follows:

#### **2.1 The denaturation**

It is the separation of the two strands of DNA, obtained by raising the temperature. The first period is carried out at a temperature of 94°C, called the denaturation

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

temperature. At this temperature, the matrix DNA, which serves as matrix during the replication, is denatured: the hydrogen bonds cannot be maintained at a temperature higher than 80°C and the double-stranded DNA is denatured into single-

The second step is hybridization. It is carried out at a temperature generally between 40 and 70°C, called primer hybridization temperature. Decreasing the temperature allows the hydrogen bonds to reform and thus the complementary strands to hybridize. The primers, short single-strand sequences complementary to regions that flank the DNA to be amplified, hybridize more easily than long strand matrix DNA. The higher the hybridization temperature, the more selective the

The third period is carried out at a temperature of 72°C, called elongation temperature. It is the synthesis of the complementary strand. At 72°C, Taq polymerase binds to primed single-stranded DNAs and catalyzes replication using the deoxyribonucleoside triphosphates present in the reaction mixture. The regions of the template DNA downstream of the primers are thus selectively synthesized. In the next cycle, the fragments synthesized in the previous cycle are in turn matrix and after a few cycles, the predominant species corresponds to the DNA sequence between the regions where the primers hybridize. It takes 20–40 cycles to synthesize an analyzable amount of DNA (about 0.1 μg). Each cycle theoretically doubles the amount of DNA present in the previous cycle. It is recommended to add a final cycle of elongation at 72°C, especially when the sequence of interest is large (greater than 1 kilobase), at a rate of 2 minutes per kilobase [1–3]. PCR makes it possible to amplify sequences whose size is less than 6 kilobases. The PCR reaction is extremely

To achieve selective amplification of nucleotide sequences from a DNA extract by PCR, it is essential to have least one pair of oligonucleotides. These oligonucleotides, which will serve as primers for replication, are synthesized chemically and must be the best possible complementarity with both ends of the sequence of interest that one wishes to amplify. One of the primers is designed to recognize complementarily a sequence located upstream of the fragment 5′–3′ strand DNA of interest; the other to recognize, always by complementarity, a sequence located upstream complementary strand (3′–5′) of the same fragment DNA. Primers are single-stranded DNAs whose hybridization on sequences flanking the sequence of interest will allow its replication so selective. The size of the primers is usually between 10 and 30 nucleotides in order to guarantee a sufficiently specific hybrid-

DNA polymerase allows replication. We use a DNA polymerase purified or cloned from of an extremophilic bacterium, *Thermus aquaticus*, which lives in hot springs and resists temperatures above 100°C. This polymerase (Taq polymerase) has the characteristic remarkable to withstand temperatures of around 100°C,

rapid, it lasts only a few hours (2–3 hours for a PCR of 30 cycles).

ization on the sequences of interest of the matrix DNA [1–5].

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

stranded DNA (single-stranded DNA).

hybridization, the more specific it is.

**2.2 Hybridization**

**2.3 Elongation**

**2.4 Primers**

**2.5 Taq polymerase**

temperature. At this temperature, the matrix DNA, which serves as matrix during the replication, is denatured: the hydrogen bonds cannot be maintained at a temperature higher than 80°C and the double-stranded DNA is denatured into singlestranded DNA (single-stranded DNA).
