**2.2 Hybridization**

*Synthetic Biology - New Interdisciplinary Science*

to purity.

**2. Principle of the PCR**

exponential amplification is achieved [1].

(the DNA of interest) will be very big. PCR makes it possible to amplify a signal from a background noise, so it is a molecular cloning method, and clone comes back

real-time PCR, competitive PCR, PCR in situ, RT-PCR, etc.

There are many applications of PCR. It is a technique now essential in cellular and molecular biology. It permits, especially in a few hours, the "acellular cloning" of a DNA fragment through an automated system, which usually takes several days with standard techniques of molecular cloning. On the other hand, PCR is widely used for diagnostic purposes to detect the presence of a specific DNA sequence of this or that organism in a biological fluid. It is also used to make genetic fingerprints, whether it is the genetic identification of a person in the context of a judicial inquiry, or the identification of animal varieties, plant, or microbial for food quality testing, diagnostics, or varietal selection. PCR is still essential for performing sequencing or site-directed mutagenesis. Finally, there are variants of PCR such as

At present, the revolutionary evolutions of the molecular biological research are based on the PCR technique which provides the suitable and specific products especially in the field of the characterization and the conservation of the genetic diversity. Several applications are possible in downstream of the PCR technique: (1) the establishment of a complete sequence of the genome of the most important livestock breeds; (2) development of a technology measuring scattered polymorphisms at loci throughout the genome (e.g., SNP detection methods); and (3) the development of a microarray technology to measure gene transcription on a large scale. The study of biological complexity is a new frontier that requires high throughput molecular technology, high speed and computer memory, new approaches to data analysis, and the integration of interdisciplinary skills.

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

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

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

process of the PCR is subdivided into three stages as follows:

**148**

**2.1 The denaturation**

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 hybridization, the more specific it is.
