Polymerase chain reaction

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Polymerase chain reaction is a technique used in molecular biology to strengthen one copy or multiple copies of a segment of DNA in multiple magnitude orders, generating thousands to millions of copies of specific DNA sequences. Developed in 1983 by Kary Mullis, an employee of Cetus Corporation, and also a Nobel Prize winner in Chemistry in 1993, this is an easy, inexpensive, and reliable way to replicate repeatedly the segment of DNA focus, a concept that applies to various fields in modern biology and related sciences. PCR is probably the most widely used technique in molecular biology. This technique is used in biomedical research, criminal forensics, and molecular archeology.

PCR is now a common and often indispensable technique used in clinical laboratories and research for a wide range of applications. These include DNA cloning for sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based phylogeny, or functional gene analysis; diagnosis and monitoring of hereditary diseases; ancient DNA amplification; analysis of genetic fingerprints for DNA profiles (for example, in forensic science and inheritance testing); and pathogen detection in nucleic acid tests for the diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.

Most PCR methods depend on the thermal cycle, which involves exposing the reactants to recurrent heating and cooling cycles, allowing various reactions to be temperature dependent - in particular, melting DNA and enzyme-driven DNA replication - to be processed many times in sequence. Primers (short DNA fragments) containing sequences that complement the target area, together with the DNA polymerase (eg Taq polymerase), after which the method is named, allowing selective and repeated amplification. When PCR takes place, the resulting DNA itself is used as a template for replication, with the regulation of chain reaction movements in which the original DNA frame is exponentially amplified. The simplicity of the basic principles underlying PCR means it can be broadly modified to perform a variety of genetic manipulations. PCR is generally not considered a recombinant DNA method, since it does not involve cutting and injecting DNA, only the amplification of existing sequences.

Almost all PCR applications use heat-resistant DNA polymerases, such as Taq polymerase, an enzyme that was originally isolated from the thermophilic bacterium Thermus aquaticus . If a heat-susceptible polymerase DNA is used, it will change every cycle in the denaturation step. Before the use of Taq polymerase, DNA polymerase must be added manually each cycle, which is a tedious and expensive process. This enzymatic protein DNA builds up new strands of DNA from free nucleotides, the building block of DNA, using single-stranded DNA as template and DNA oligonucleotides (mentioned above) to begin DNA synthesis.

In the first step, two double helical strands of DNA are physically separated at high temperatures in a process called melted DNA. In the second step, the temperature is lowered and the two DNA strands become the template for the DNA polymerase to selectively amplify the target DNA. Selectivity of PCR results from primary use that complements the sequence around the targeted DNA region for amplification under special thermal cycling conditions.

PCRs, such as recombinant DNA technology, have enormous impacts in both the basic and molecular biological aspects of the molecular biology as they can produce a large number of specific DNA fragments from a small number of complex templates. Recombinant DNA techniques create clones of molecules by conferring on a specific sequence the ability to replicate by inserting them into vectors and introducing vectors into the host cell. The PCR represents the "in vitro" cloning form that can produce, as well as modify, the DNA fragment of the length and sequence specified in a simple automatic reaction. In addition to its many applications in basic molecular biology research, PCR pledges to play an important role in the identification of important medical sequences as well as an important diagnostic in their detection.

Video Polymerase chain reaction

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PCR amplifies specific regions of DNA strands (DNA targets). Most PCR methods amplify DNA fragments between 0.1 and 10 kilo base pairs (kbp), although some techniques allow for the amplification of fragments up to 40 kbp. The amount of reinforced product is determined by the substrate available in the reaction, which becomes limited when the reaction takes place.

Basic PCR settings require several components and reagents, including:

• a DNA template containing the target area of ​​DNA to strengthen
• a DNA polymerase , an enzyme that polymerizes new strands of DNA; thermal resistance Taq polymerase is very common, as it is more likely to remain intact during high temperature DNA denaturation processes
• two primary DNAs complementing the 3 '(three base) ends of each senses and the anti-sense strands of the DNA target (DNA polymerases can only bind and extend from the double-stranded region of DNA ; without a primary there is no double-stranded initiation site where the polymerase can bind); specific primers that complement selected target DNA areas, and are often custom-made in laboratories or purchased from commercial biochemical suppliers.
• tripophosphate deoxynucleoside , or dNTPs (sometimes called "tripophosphate deoxynucleotide"; nucleotides containing triphosphate groups), the building blocks from which DNA polymerases synthesize new strands of DNA
• buffer solution provides an appropriate chemical environment for optimal activity and stability of the DNA polymerase
• bivalent cations , usually magnesium (Mg) or manganese (Mn) ions; Mg ² is the most common, but Mn ² can be used for PCR-mediated DNA mutagenesis, as higher concentration of Mn ² increases the error rate during DNA synthesis
• monovalent cations , usually potassium ions (K)

Reactions are generally done in volumes 10-200? L in a small reaction tube (0.2-0.5 ml volume) in a thermal cyclist. The thermal cyclist heats and cools the test tube to reach the required temperature at each reaction step (see below). Many modern thermal cyclists use Peltier effect, which allows heating and cooling blocks that hold PCR tubes by simply reversing the electric current. The thin-walled reaction tube allows favorable thermal conductivity to allow rapid thermal equilibrium. Most thermal cyclers have a heating cap to prevent condensation at the top of the test tube. Older thermal cyclers that do not have a heated cover require a layer of oil over the reaction mixture or a wax ball inside the tube.

Procedures

To check whether PCR succeeds in generating the anticipated target area of ​​DNA (also sometimes referred to as amplimer or amplicon), agarose gel electrophoresis can be used for separation of PCR product sizes. The PCR product size is determined by comparison with the DNA ladder, the molecular weight marker containing the DNA fragment of a known size running on the gel next to the PCR product.

Stages

Like other chemical reactions, the reaction rate and efficiency of PCR are influenced by limiting factors. Thus, the entire PCR process can then be divided into three stages based on the progress of the reaction:

• Exponential gain : In each cycle, the number of products is duplicated (assuming 100% reaction efficiency). After 30 cycles, one copy of DNA can be increased up to 1 000 000 copies. In a sense, then, the replication of a discrete strand of DNA is manipulated in a tube under controlled conditions. The reaction is very sensitive: only a small quantity of DNA must exist.
• Leveling off stage : The reaction slows down when the DNA polymerase loses activity and because the consumption of reagents such as dNTP and primary causes them to be limited.
• Plateau : No more products accumulate due to fatigue of reagents and enzymes.

Maps Polymerase chain reaction

Optimization

In practice, PCR can fail for various reasons, partly because of its sensitivity to contamination that causes the amplification of fake DNA products. Therefore, a number of techniques and procedures have been developed to optimize PCR conditions. Contamination with foreign DNA is treated with laboratory protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves the spatial separation of PCR regulatory areas from the area for analysis or purification of PCR products, use of disposable plasticware, and thoroughly clean the working surface between reaction settings. Primary design techniques are important in improving the yield of PCR products and in avoiding the formation of counterfeit products, and the use of alternative buffer components or polymerase enzymes may help with amplification of long or problematic DNA areas. The addition of reagents, such as formamide, in a buffer system can increase the specificity and yield of PCR. Computer simulation of theoretical PCR results (Electronic PCR) can be done to assist in the primary design.

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Apps

Selective DNA isolation

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of certain DNA regions. The use of PCR adds many ways, such as generating hybridization probes for southern or northern hybridization and DNA cloning, requiring larger amounts of DNA, representing specific DNA regions. PCR supplies these techniques with a high amount of pure DNA, allowing analysis of DNA samples even from very few starting materials.

Other PCR applications include DNA sequencing to determine the unknown sequence of PCR-amplification in which one amplifier primer can be used in Sanger sequencing, isolating DNA sequences to accelerate recombinant DNA technology involving insertion of DNA sequences into plasmids, phages, or cosmids (depending on on size) or genetic material from other organisms. Bacterial colonies (such as E. coli) can be quickly filtered by PCR for the construction of the correct DNA vector. PCR can also be used for genetic fingerprinting; a forensic technique used to identify a person or an organism by comparing experimental DNA through different PCR-based methods.

Some PCR fingerprint methods have high discriminatory powers and can be used to identify genetic relationships between individuals, such as parent-child or siblings, and are used in paternity testing (Figure 4). This technique can also be used to determine the evolutionary relationship between organisms when certain molecular clocks are used (ie, 16S rRNA and recA gene from microorganisms).

Amplification and DNA quantification

Because PCR amplifies the target DNA area, PCR can be used to analyze small samples. This is often important for forensic analysis, when only a small amount of DNA is available as evidence. PCR can also be used in ancient DNA analyzes that are tens of thousands of years old. These PCR-based techniques have been successfully used in animals, such as forty thousand-year-old mammals, as well as human DNA, in applications ranging from Egyptian mummified analysis to the identification of the Czar tsar and the human body. King of England Richard III.

Quantitative PCR or Real Time Quantitative PCR (RT-qPCR) methods allow estimation of the number of given sequences present in the sample - a technique often applied to determine the quantity of gene expression quantitatively. Quantitative PCR is an established tool for DNA quantification that measures the accumulation of DNA products after each round of PCR amplification.

RT-qPCR allows the quantification and detection of specific DNA sequences in real time as it measures the concentration during the synthesis process. There are two methods for simultaneous detection and quantification. The first method consists of using a fluorescent dye that is maintained not specifically among the double strands. The second method involves probing the code for a specific order and being labeled fluorescently. DNA detection using this method can only be seen after hybridization of the probe with complementary DNA occurs. The combination of interesting techniques is real-time PCR and reverse transcription. This advanced technique allows the quantification of small amounts of RNA. Through this combined technique, the mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the likelihood of error at the PCR endpoint, increasing the chances of detecting genes associated with genetic diseases such as cancer. The laboratory uses RT-qPCR for the purpose of sensitively measuring gene regulation.

Medical applications

. Prospective parents can be tested to be genetic carriers, or their children may be tested to actually get sick. DNA samples for prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by analysis of rare fetal cells circulating in the maternal bloodstream. PCR analysis is also important for preimplantation genetic diagnosis, in which individual cells of developing embryos are tested for mutations.

• PCR can also be used as part of a sensitive test for typing network , essential for organ transplants. In 2008, there were even proposals to replace traditional antibody-based tests for blood type with PCR-based assays.
• Many forms of cancer involve changes to oncogenes . Using PCR-based tests to study these mutations, treatment regimens can sometimes be individually adjusted for the patient. PCR allows the early diagnosis of malignant diseases such as leukemia and lymphoma, which are currently highest in cancer research and are already used routinely. PCR tests can be performed directly on genomic DNA samples to detect malignant translocation-specific cells at sensitivity that are at least 10,000 times higher than other methods. PCR is particularly useful in the medical field as it allows for the isolation and amplification of tumor suppressors. Quantitative PCR, for example, can be used to measure and analyze single cells, as well as recognize DNA, mRNA and confirmation and combination of proteins.

Application of infectious diseases

PCR allows the rapid and very specific diagnosis of infectious diseases, including those caused by bacteria or viruses. PCR also allows identification of non-developmentable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture and animal modeling tests. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and non-pathogenic discrimination of pathogen strains based on specific genes.

Characterization and detection of infectious disease organisms has been revolutionized by PCR in the following ways:

• The human immunodeficiency virus (or HIV ) is a difficult target to discover and eradicate. The earliest test for infection depends on the presence of antibodies against the virus circulating in the bloodstream. However, antibodies do not appear until several weeks after infection, maternal antibodies covering infections in newborns, and therapeutic agents to fight infections do not affect antibodies. A PCR test has been developed that can detect at least one viral genome between the DNA of more than 50,000 host cells. Infection can be detected early, donated blood can be filtered directly for the virus, newborns can be tested for infection immediately, and the effects of antiviral treatment can be quantified.
• Some disease organisms, such as for tuberculosis , are difficult to sample from patients and are slow to be planted in the laboratory. PCR-based testing has enabled the detection of a small number of disease organisms (either live or dead), in appropriate samples. Detailed genetic analysis can also be used to detect antibiotic resistance, which allows immediate and effective therapy. Therapeutic effects can also be evaluated immediately.
• The spread of disease organisms through domestic or wild animal populations can be monitored by PCR testing. In many cases, the emergence of a vicious new sub-type can be detected and monitored. Sub-types of organisms responsible for previous epidemics can also be determined by PCR analysis.
• Viral DNA can be detected by PCR. The primer used should be specific to the targeted sequence in viral DNA, and PCR can be used for diagnostic analysis or DNA sequencing of the viral genome. High sensitivity of PCR allows detection of the virus immediately after infection and even before the onset of the disease. Such early detection can give doctors significant waiting time in treatment. Viral load ("viral load") in patients can also be quantified by PCR-based DNA quantization techniques (see below).
• Diseases such as pertussis (or whooping cough) are caused by Bordetella pertussis bacteria. These bacteria are characterized by serious acute respiratory infections that affect various animals and humans and have caused the death of many small children. Pertussis toxin is a protein exotoxin that binds cell receptors by two dimers and reacts with different cell types such as T lymphocytes that play a role in cell immunity. PCR is an important testing tool that can detect sequences in pertussis toxin genes. This is because PCR has a high sensitivity to toxins and has shown a fast turnaround time. PCR is very efficient to diagnose pertussis when compared with culture.

Forensics app

Development of PCR-based genetic (or DNA) fingerprint protocols has been widely used in forensics:

• In its most discriminating form, genetic fingerprint can uniquely distinguish one person from the rest of the world's population. A small number of DNA samples can be isolated from a crime scene, and compared with a sample from a suspect, or from a DNA database of previous evidence or prisoners. A simpler version of the test is often used to quickly remove a suspect during a criminal investigation. Evidence of decades-old crimes can be tested, confirmed or acquitted of persons previously convicted.
• Typing forensic DNA has become an effective way to identify or free criminal suspects because of the evidence analysis found at the scene. The human genome has many recurring regions that can be found in gene sequences or in areas of non-encoding genomes. In particular, up to 40% of human DNA recurs. There are two different categories for these repeating non-coding areas in the genome. The first category is called the variable number tandem repeats (VNTR), which is 10-100 long base pairs and the second category is called short tandem repetition (STR) and it consists of 2-10 pair of base pairs repeated. PCR is used to strengthen some of the famous VNTRs and STRs using primers flanking each recurring region. The size of fragments obtained from each individual for each STR will show which alleles exist. By analyzing multiple STRs for individuals, a set of alleles for each person will be found statistically likely to be unique. Researchers have identified the complete sequence of the human genome. This sequence can be easily accessed through the NCBI website and is used in many real-life applications. For example, the FBI has collected a set of DNA marker sites used for identification, and this is called DNA Combined DNA System database (CODIS). Using this database allows statistical analysis to be used to determine the probability that a DNA sample would match. PCR is a very powerful and significant analytical tool for use for typing forensic DNA because researchers require only a small amount of target DNA to be used for analysis. For example, one piece of human hair with an attached hair follicle has enough DNA to perform the analysis. Similarly, some sperm, skin samples from under the nail, or small amounts of blood can provide enough DNA for conclusive analysis.
• The less discriminatory form of DNA fingerprints can be helpful in DNA DNA testing , in which an individual is matched to a close relative. DNA from unidentified human remains can be tested, and compared to the possibilities of parents, siblings, or children. Similar tests may be used to confirm the biological parents of the adopted (or kidnapped) child. The true biological father of a newborn can also be confirmed (or ruled out).
• The AMGX/AMGY PCR design has proven not only to facilitate the strengthening of DNA sequences from very small number of genomes. However it can also be used for the determination of real time sex from forensic bone samples. This gives us a powerful and effective way to determine the sexes not only of ancient specimens but also of suspects in crime.

The research application

PCR has been applied to many areas of research in molecular genetics:

• PCR allows the rapid production of short DNA pieces, even when no more than the order of two known primers. This PCR capability adds many methods, such as producing a hybridized probe for Southern or Northern blot hybridization. PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, allowing analysis even from the very small amount of starting material.
• The DNA sequencing task can also be aided by PCR. Known DNA segments can be easily generated from patients with mutations of genetic diseases. Modifications to amplification techniques can extract segments from a completely unknown genome, or can produce just one strand of an area of ​​interest.
• PCR has many applications for more traditional DNA cloning processes. It can extract segments to be inserted into vectors of the larger genome, which may be available only in small quantities. Using a set of 'primary vectors', it can also analyze or extract fragments that have been inserted into the vector. Some changes to the PCR protocol can generate mutations (in general or by site) of the inserted fragments.
• Serial consecutive sites is the process by which PCR is used as an indicator that certain segments of the genome are present in certain clones. The Human Genome Project finds this application important to map their sorted cosmid clones, and coordinate results from different laboratories.
• The exciting application of PCR is the philogenic analysis of DNA from ancient sources , such as those found in restored Neanderthal bones, from frozen mammoth tissue, or from Egyptian mummified brains. It has been strengthened and sorted. In some cases, highly degraded DNA from these sources can be reassembled during the initial stage of amplification.
• The general application of PCR is the study of gene expression patterns . Networks (or even individual cells) can be analyzed at different stages to see which genes are active, or that have been turned off. This application can also use quantitative PCR to measure actual expression levels
• The ability of PCR to simultaneously strengthen several loci from individual sperm has greatly enhanced the more traditional task of genetic mapping by studying crossover chromosomes after meiosis. A rare crossover event between very close loci has been observed directly by analyzing thousands of individual sperm. Similarly, unusual deletion, insertion, translocation, or inversion can be analyzed, all without having to wait (or pay) for a long and tiring fertilization process, embryogenesis, etc.

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Advantages

PCR has a number of advantages. It's fairly easy to understand and use, and produces results quickly. This technique is very sensitive with the potential to generate millions to billions of copies of certain products for sorting, cloning, and analyzing. qRT-PCR shares the same advantages as PCR, with the added advantage of quantifying synthesized products. Therefore, it has the utility to analyze changes in the level of gene expression in tumors, microbes, or other disease states.

PCR is a very powerful and practical research tool. Unknown aetiological sequences of many diseases are being sought by PCR. This technique can help identify previously unknown virus sequences related to the already known and thus give us a better understanding of the disease itself. If this procedure can be further simplified and sensitive non-radiometric detection systems can be developed, PCR will take prominent places in clinical laboratories for years to come.

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Limitations

One of the major limitations of PCR is that prior information about the target sequence is required to produce a primer that will allow selective amplification. This means that, usually, PCR users must know the exact (upstream) sequence of the target region on each of the two single-stranded templates to ensure that the DNA polymerase binds properly to the primary template hybrid and then generates the entire target area during DNA synthesis.

Like all enzymes, DNA polymerases are also susceptible to errors, which in turn lead to mutations in the resulting PCR fragments.

A 1971 paper in the Journal of Molecular Biology by Kjell Kleppe and colleagues in the laboratory H. Gobind Khorana first described methods using enzymatic assays to replicate short DNA frames with primers in vitro >. However, the early manifestations of this basic PCR principle did not receive much attention at the time, and the discovery of the polymerase chain reaction in 1983 was generally credited to Kary Mullis.

When Mullis developed PCR in 1983, he worked at Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing a short chain of DNA. Mullis has written that he contains PCR while sailing along the Pacific Coast Highway one night in his car. He plays in his mind in a new way of analyzing the changes (mutations) in DNA when he realizes that he instead finds methods of strengthening the area of ​​DNA through a repetitive cycle of duplication that is driven by DNA polymerase. In Scientific American, Mullis summarizes the procedure: "Starting with a DNA molecule of genetic material, PCR can produce 100 billion similar molecules in the afternoon.This reaction is easy to execute.This requires no more than a test tube, some reagents simple, and heat sources. "The DNA fingerprint was first used for paternity testing in 1988.

Mullis was awarded the Nobel Prize in Chemistry in 1993 for his discovery, seven years after he and his colleagues at Cetus first proposed his proposal for practice. Mullis's 1985 paper with RK Saiki and HA Erlich, "Enzymatic Amplification of Genomic -globin Sequence and Site Analysis Restriction for Diagnosis of Sickle Cell Anemia" - discovery of polymerase chain reaction (PCR) - respected by the Citation for Chemical Breakthrough Award from the Division of the Chemical History of the Chemical Society America by 2017.

Some controversy remains about the intellectual and practical contributions of other scientists for Mullis's work, and whether he has been the sole inventor of the principle of PCR (see below).

In essence the PCR method is the use of an appropriate DNA polymer capable of withstanding high temperatures & gt; 90 Â ° C (194 Â ° F) is required for the separation of two DNA strands in a double helix of DNA after each replication cycle. The DNAase polymers initially used for in vitro experiments using PCR were unable to withstand this high temperature. So the initial procedure for DNA replication is very inefficient and time-consuming, and requires a large amount of DNA polymerase and continuous handling during the process.

The discovery in 1976 of polymerase Taq - polymerase DNA purified from thermophilic bacteria, Thermus aquaticus , which naturally lives in hot environments (50 to 80 ° C (122 to 176 ° F)) such as hot springs - paving the way for dramatic improvements to PCR methods. DNA polymerase isolated from T. aquaticus is stable at elevated temperatures active even after DNA denaturation, thus negating the need to add new DNA polymerases after each cycle. This enables an automated thermocycler-based process for DNA amplification.

Patent disputes

The PCR technique was patented by Kary Mullis and assigned to Cetus Corporation, where Mullis worked when he discovered the technique in 1983. The polymerase enzyme Taq is also protected by patents. There are several high profile lawsuits related to the technique, including a failed lawsuit filed by DuPont. Hoffmann-La Roche pharmaceutical company purchased a patent in 1992 and currently holds the rights that are still protected.

The related patent battles on the Taq polymerase enzyme still take place in several jurisdictions around the world between Roche and Promega. The legal argument has surpassed the life of the original PCR and Taq polymerase patent, which ended on March 28, 2005.

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See also

• Amplified isothermal loop-mediated
• spiking DNA

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References

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External links

• Guide to PCR Technologies SelectScience
• PCR Animation maxanim.com
• OpenPCR PCc Project open source thermalcycler
• US patent for PCR
• Step-through animation of PCR - Cold Spring Harbor Laboratory
• OpenWetWare
• What is the plateau PCR effect? YouTube tutorial videos
• Primary PCR GeneWarrior Online design tool
• History of Polymerase Chain Reaction from Smithsonian Institution Archives
• 3d model of PCR equipment for 3D printing on thingiverse.com
• Computer training. PCR and PCR-RFLP experiment design

Source of the article : Wikipedia