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Genes and Genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from nucleotides by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, that is, alternately heating and cooling the PCR sample in a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of the PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions (Figure 2.17).
Exploration of Extremophiles for Value-Added Products
Published in Pratibha Dheeran, Sachin Kumar, Extremophiles, 2022
Surojit Bera, Trinetra Mukherjee, Subhabrata Das, Sandip Mondal, Suprabhat Mukherjee, Sagnik Chakraborty
Extremophiles are rich sources of biomolecules with diverse applications in biotechnology, nutrition, veterinary medicine, and human medicine. The best-studied and well-known are enzymes, particularly thermophile enzymes and, increasingly, psychrophiles, which are used in applications of all kinds in molecular biology. DNA polymerase from thermophiles, a cornerstone of PCR-based diagnostics for a wide variety of animal pathogens, is the most commonly recognized use of an extremophile drug in pharmaceutics. Thermophilic eubacterial microorganism Thermus aquaticus produces a thermostable DNA polymerase enzyme called Taq polymerase. These enzymes can withstand at 50—80°C, thus allowing the development of a polymerase chain reaction (PCR) in which double-stranded DNA is denatured at temperatures > 90°C and copied using DNA polymerase (Ishino and Ishino 2014). The creation of various forms of PCR and the availability of a variety of extremophile DNA polymerases has revolutionized human diagnostics and has allowed researchers to reveal comprehensive genomic information on many domestic and wild animal species. Real-time PCR has improved and simplified laboratory methods and has allowed researchers and clinicians to obtain more knowledge from laboratory-submitted specimens. Many significant animal viruses are detected using PCR (RT—PCR) reverse transcription technologies, including foot-and-mouth, swine fever, bluetongue, and the avian influenza virus and Newcastle disease virus (Hoffmann et al. 2009). PCR-based approaches are also used to identify associated animal viruses, equine parasites, new aquatic animal diagnostics, and parasite detection.
Molecular Analysis in Mechanobiology
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
PCR was invented by Dr. Kary Mullis and colleagues at Cetus Corp in the mid-1980s [26,27]. The reaction consists of a template DNA molecule, two short DNA oligonucleotides complimentary to the template (primers), DNA polymerase, buffer with appropriate Mg2+ concentration, and dNTPs. Through a process of thermal cycling, a specific region of the DNA template is selectively amplified. The use of a thermostable DNA polymerase (Taq polymerase) derived from the bacterium Thermus aquaticus substantially improved the efficiency and utility of PCR [28]. Reverse transcriptase, an RNA-dependent DNA polymerase that synthesizes cDNA from an RNA template allowed the application of PCR to mRNA analysis (RT-PCR).
Identification of microbes from textile dye wastewater and its antibiotic resistance from local textile factory
Published in Bioremediation Journal, 2023
Nur Hanis Mohamad Hanapi, Hadieh Monajemi, Azimah Ismail, Zarizal Suhaili, Hafizan Juahir
In a PCR synthesis, there are a few important components that are needed to be in the PCR reaction mixtures for the reaction to take place. As listed in Table 1, the important components are 10x PCR reaction buffer, 10µM of forward primer (27f, AGAGTTTGATCMTGGCTCAG), 10µM of reverse primer (1492r, GGTTACCTTGTTACGACTT),10µM of dNTPs mix, Taq polymerase, 25µM of MgCl2, ultra-pure water and the DNA extract that were extracted from the previous extraction methods. The dNTPs, or deoxyribonucleoside triphosphates, is one of the vital components in the synthesis of PCR. Each dNTPs is made up of a phosphate group, which is a deoxyribose sugar and a nitrogenous base, and there are purine and pyrimidine groups that consist of two bases each. Unbalanced or insufficient amounts of dNTPs can cause serious genotoxic consequences (Chen et al. 2014). Taq polymerase, has been explained briefly as a polymerase enzyme which was isolated from Thermus aquaticus, indicating the ability to stand high temperatures thus it is very appropriate to be applied in the denaturation stage (Johnson 1991). Another component is MgCl2 where it plays a vital contribution in the process. During the PCR synthesis, MgCl2 acts as a cofactor and catalyzer. The concentration of MgCl2 will affect the Taq polymerase activities in synthesizing the DNA sequence. Study by Cobb and Clarkson (1994) presented the findings on the different MgCl2 concentration affecting the size of PCR amplification products, making it very important in PCR synthesis.