A polymerase chain reaction is a technique that has been there for more than 20 years. It is recognized as one of the most important scientific advances in the 20th century. This innovation has a broad variety of applications. I have chosen PCR as my topic of discussion because of its wide range of applications. It is in so doing that can to apply the above technique in medical and diagnostics, infectious diseases, forensic application, research application.
Keywords: polymerase chain reaction; PCR
People behind the innovation.
In 1983, Kary Mullis invented the PCR technique. He was a scientist at the Cetus Corporation, conceived of PCR as a method to copy DNA and synthesize large amounts of a specific target DNA. Following years, a team of scientists at Cetus Corporation recognized the potential impact PCR could have on molecular biology, researched, refined and, made the theoretical process a reality.
The team presented for the first time in 1985 at the American Society for Human Genetics annual meeting. Two significant advances have enabled PCR to become the technology it is today: Taq polymerase and the thermal cycler.
Series of events that led to innovation.
Following the invention of the polymerase chain reaction, there were significant advances that included Taq polymerase and thermal cycler. Taq polymerase was isolated in 1986 by the Cetu scientists from the Thermus aquaticus which is a bacterium. Taq could not withstand high temperatures therefore, eliminating the need for human intervention during the reaction. This streamlined and shortened the process. The process would be cumbersome without a heat resistant enzyme-like Taq polymerase if used on a large scale process. PerkinElmer launched a thermal cycler in 1987. It’s an instrument that is programmed to regulate the temperature of a reaction, heating or, cooling the sample as needed. In this process, human interaction was minimized. This led to an elegant, efficient and streamlined process.
The process of polymerase chain reaction is as follows:
Initialization is only required for DNA polymerases that require heat activation by hot-start PCR.
Denaturation is the first regular cycling event and consists of heating the reaction chamber to 94–98 °C (201–208 °F) for 20–30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules.
Annealing is the next step. the reaction temperature is lowered to 50–65 °C (122–149 °F) for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Determination of proper temperature for this step is important because efficiency and specificity are strongly affected b the annealing temperature. hybridization is achieved by the low temperatures but also high enough to ensure hybridization is specific. In this step, polymerase binds to the primer-template hybrid beginning the process of DNA formation.
Extension/elongation: Temperature at this step depends on the DNA polymerase used. A new DNA strand that is complementary to the DNA template is synthesized by DNA polymerase by adding free dNTPs from the reaction mixture that is complementary to the template. Apart from the DNA polymerase used, the elongation process also depends on the length of the target DNA region to amplify. Denaturation, annealing, and elongation constitute a single cycle. Therefore multiple cycle s are required to amplify DNA.
The final step cools the reaction chamber to 4–15 °C for an indefinite time and maybe employed for short-term storage of the PCR products.
Medical and diagnostic applications.
Parents can be tested as genetic carriers or their children may be tested for being affected by a certain disease. DNA samples obtained by amniocentesis, chorionic villus sampling for prenatal testing, or analysis of rare fetal cells circulating in the mother’s bloodstream. PCR analysis is also essential to preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations.
PCR is also used as a sensitive test for tissue typing which is vital to organ transplantation.
Using PCR based tests to study alterations in cancer oncogenes, therapy regimens can be customized to a patient. It permits early diagnosis of malignant diseases such as leukemias and lymphomas. PCR is very useful in the medical field since it allows for the isolation and amplification of tumor suppressors.
Infectious disease applications.
PCR allows for rapid and highly specific diagnoses of infectious diseases, such as those caused by bacteria or viruses. PCR also permits the identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models.
Human immunodeficiency virus is a difficult virus to find and eradicate. PCR tests have made it possible to detect infections earlier. this is because as little as one viral genome among DNA of many host cells can be detected. Newborns can be tested for infection and the effects of antiviral treatments quantified.
Disease organisms such as that for tuberculosis are slow to be grown in laboratories. Hence PCR based tests allow for the detection of a small number of organisms in obtained samples. Genetic analysis can also be used to detect antibiotic resistance allowing immediate and effective therapy.
Increased Detection of Viruses in Children with Respiratory Tract Infection Using PCR
Respiratory viruses are a common cause of respiratory tract infection, especially in neonates and children. Accurate diagnosis improved clinical outcomes and reduce the use of antibiotics. Real-time PCR is better than traditional antigen tests and virus cultures when considering the detection of respiratory viruses.
Application and Optimization of RT-PCR in the Diagnosis of SARS-CoV-2 Infection
Coronavirus Disease 2019 (COVID-19) caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global threat to public health. The diagnostic accuracy of the pharyngeal swab RT-PCR, CT, combined with the second pharyngeal swab RT-PCR or with CT were evaluated individually. RT-PCR is superior to CT in diagnosing mild infections. Stool RT-PCR should be considered as an item for improving discovery rate and hospital discharge. This study sheds light on optimizing the scheme of screening and monitoring of SARS-CoV-2 infection.
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