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. It only takes 2–3 hours to get a billion or so copies.The characterization of the diversity of species living within ecosystems is of major scientific interest to understand the functioning of these ecosystems. The cycle is repeated many times (usually 20–30) as most processes using PCR need large quantities of DNA. The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand.
The order in which the free nucleotides are added is determined by the sequence of nucleotides in the original (template) DNA strand. This enzyme is often Taq polymerase, an enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus. A DNA polymerase enzyme joins free DNA nucleotides together. New strands of DNA are made using the original strands as templates. This can only occur once the temperature of the solution has been lowered. Primers bind to the target DNA sequences and initiate polymerisation. The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break. As in DNA replication, the two strands in the DNA double helix need to be separated.