PCR makes it possible to obtain, by in vitro replication, multiple copies of a DNA fragment from an extract. 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.
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. Finally, there are variants of PCR such as real-time PCR, competitive PCR, PCR in situ, RT-PCR, etc.Īt 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. PCR is still essential for performing sequencing or site-directed mutagenesis. 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. 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 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. There are many applications of PCR. It is a technique now essential in cellular and molecular biology. PCR makes it possible to amplify a signal from a background noise, so it is a molecular cloning method, and clone comes back to purity. In contrast, the amount of the amplified sequence(s) (the DNA of interest) will be very big.
Once the reaction is complete, the amount of matrix DNA that is not in the area of interest will not have varied. From such a mass of sequences that constitutes the matrix DNA, the PCR can therefore select one or more sequences and amplify them by replication to tens of billions of copies. It is therefore necessary to isolate and purify the sequence or sequences that are of interest, whether it is the sequence of a gene or noncoding sequences (introns, transposons, mini or microsatellites). It contains many mass of nucleotide sequences. DNA extracted from an organism or sample containing DNAs of various origins is not directly analyzable.
PCR is therefore a technique of purification or cloning. We can therefore amplify nucleotide sequences from infinitesimal amounts of DNA extract. The power of PCR is based on the fact that the amount of matrix DNA is not, in theory, a limiting factor. Indeed, if the sequence of interest is present in the DNA extract, it is possible to selectively replicate it (we speak of amplification) in very large numbers. This method can generate tens of billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA template). Its principle is based on the use of DNA polymerase which is an in vitro replication of specific DNA sequences. Polymerase chain reaction (PCR) was invented by Mullis in 1983 and patented in 1985. The study of biological complexity is a new frontier that requires high-throughput molecular technology, high speed computer memory, new approaches to data analysis, and the integration of interdisciplinary skills.
In genetic diversity studies, the most frequently used markers are microsatellites. A number of markers are now available to detect nuclear DNA polymorphisms. The need to understand the molecular mechanisms in species has made the PCR an indispensable tool for understanding the functioning of these biological systems. Historically, species have been described and characterized on the basis of morphological criteria, which are closely linked by environmental conditions or which find their limits especially in groups where they are difficult to access, as is the case for many species of microorganisms. It is also becoming a societal issue since it is necessary to implement the conservation or even the restoration of biodiversity. The characterization of the diversity of species living within ecosystems is of major scientific interest to understand the functioning of these ecosystems.