How is pcr used

Read What is PCR? Modern biotechnology introduces some of the ways in which DNA technologies have changed what scientists are able to do. In this interactive, learn about the diverse range of technologies that are possible because of PCR. PCR contributes to our understanding of many environmental issues, particularly where the detection of microorganisms in the environment is required. PCR allows specific target species to be identified and quantified, even when very low numbers exist.

One common example is searching for pathogens or indicator species such as coliforms in water supplies. Find out how PCR has been used by scientists to explore the environment in Developing an assay , Detecting viruses in the environment , Life in the upper troposphere and Virus hunters.

To think about: As a population, we are becoming more aware of the importance of water quality. Whose responsibility should it be to monitor water quality? Who should be allowed access to information about water quality? Whose responsibility should it be to maintain good water quality? PCR has enabled personalised genome testing. An industry has sprung up offering consumers tailor-made products and services based on information in their genome.

For example, nutrigenomics is a specific form of consumer genomics linking genetic information to information about foods that might be better or worse for particular conditions, like inflammatory bowel disease. Food for thought: How could you be sure the products are really useful? Genetic diseases and paternity testing Another important application of PCR is in the analysis of mutations that occur in many genetic diseases e. How does PCR work?

Primers: Single stranded oligonucleotides that match exactly the beginning and end of the DNA template. These are generated synthetically. These are added in excess amount. A buffer solution: Creates an optimal environment for the reaction to occur in. The first step is known as the denaturation step and is carried out at around 95 o C.

The second step is the annealing step and is carried out at about o C. Once synthesis has been completed, the whole mixture is heated again to 95 o C to melt the newly formed DNA complexes. This results in twice the amount of template available for the next round of replication. Repeated heating and cooling quickly amplifies the DNA segment of interest. Roughly one million copies are made after 20 cycles.

Molecular Biology of the Cell. The Polymerase chain reaction. Switzerland: Birkhauser Press, Human diseases caused by viruses In: Microbiology. Lodish H et al. The polymerase chain reaction: An alternative to cloning. In: Molecular Cell Biology. New York: Scientific American Books ; p. Hanscheid T. Diagnosis of malaria: A review of alternatives to conventional microscopy. Clin Lab Haem.

Hoffmaster AF et al. Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis. Emerging Infectious Diseases. Once the reaction is complete, the amount of matrix DNA that is not in the area of interest will not have varied.

In contrast, the amount of the amplified sequence s 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 to purity. There are many applications of PCR. It is a technique now essential in cellular and molecular biology.

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. 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.

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.

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 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:. It is the separation of the two strands of DNA, obtained by raising the temperature. The second step is hybridization. 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. It is the synthesis of the complementary strand. 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. Each cycle theoretically doubles the amount of DNA present in the previous cycle. PCR makes it possible to amplify sequences whose size is less than 6 kilobases.

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.

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 hybridization on the sequences of interest of the matrix DNA [ 1 , 2 , 3 , 4 , 5 ].

DNA polymerase allows replication. 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.

It is possible to add detergents Tween 20, Triton X 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 MgCl 2 [ 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.

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.

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. The amount of template DNA in the reaction medium initiate that the amplification reaction can be reduced to a single copy. In general, the amounts used are in the range of 10— 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 , 7 , 8 ]. 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 visible by ultraviolet transillumination — nm. Very small products are often visible very close to the migration front in the form of more or less diffuse bands.

They correspond to primer dimers and sometimes to the primers themselves. On automated systems, a fragment analyzer is now used. This apparatus uses the principle of capillary electrophoresis. Fragment detection is performed by a laser diode. This is only possible if the PCR is performed with primers coupled to fluorochromes [ 10 ]. 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. 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.

Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification. Often heralded as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B.

Mullis, was awarded the Nobel Prize for Chemistry in