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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).
Genes and genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Elongation step: The temperature at this step depends on the DNA polymerase used. Taq polymerase has its optimum activity temperature at 75°C–80°C, and commonly a temperature of 72°C is used with this enzyme. At this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5’–3’ direction, condensing the 5’-phosphate group of the dNTPs with the 3’-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule of thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, that is, if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Halophiles: Pharmaceutical Potential and Biotechnological Applications
Published in Devarajan Thangadurai, Jeyabalan Sangeetha, Industrial Biotechnology, 2017
Rebecca S. Thombre, Vaishnavi S. Joshi, Radhika S. Oke
With the renaissance of biotechnology, science has demanded the search of new enzymes by employing the techniques of genetic engineering or by searching a new source of enzymes (Oren, 2002). Scientists have therefore diverted their focus on the new domain called Archaea – the extremophiles. Archaea can withstand harsh and strict conditions hence their use in industries like environmental remediation, food processing, detergents industry, need such biocatalysts. Until recently the source of these enzymes have been plants, animals, fungi and microbes from bacterial domain, but as the enzymes of these organisms work efficiently at high pH, salinity, temperature, they have gained a lot of importance (Synowiecki et al., 2006; Ozcan et al., 2009). These enzymes are efficient in catalyzing the polymerase chain reaction. Taq polymerase is isolated from thermophilic bacteria is used extensively in PCR reactions. The demand for such enzymes is growing manifold and there has arisen the need to clone the genes encoding these enzymes in some other organisms (Alqueres et al., 2007).
Isolation and characterization of yogurt starter cultures from traditional yogurts and growth kinetics of selected cultures under lab-scale fermentation
Published in Preparative Biochemistry & Biotechnology, 2023
Selma Kayacan Çakmakoğlu, Melike Vurmaz, Emine Bezirci, Yasemin Kaya, Hilal Dikmen, Hamza Göktaş, Fatmanur Demirbaş, Betül Encu, Esra Acar Soykut, Filiz Alemdar, İbrahim Çakır, M. Zeki Durak, Muhammet Arıcı, Osman Sağdıç, Mustafa Türker, Enes Dertli
For the genotypic discrimination of the Lb. delbrueckii subps. bulgaricus and S. thermophilus strains, RAPD-PCR analysis using GTG5 primer were applied using the previously described methodology.[16] For this, PCR mixtures containing genomic DNA, 0.25 µL Taq polymerase (Thermo Fisher Scientific, US), 1.5 µL, 20 mM of GTG5 primer (5′-GTGGTGGTGGTGGTG-3′), 5 µL PCR buffer (Thermo Fisher Scientific, US), 4 µL, 20 mM dNTPS (Thermo Fisher Scientific, US), were prepared. The amplification was carried out with a model T100 thermal cycler (Bio-rad, US) and the RAPD-PCR was performed with the following program: 95 °C for 10 min, 35 cycles of 94 °C for 1 min, 40 °C for 1 min, 65 °C for 8 min, then final step of 65 °C for 16 min. The PCR products were separated with electrophoresis on 1% (wt/vol) agarose gels at 120 V for 1.5 hr and band patterns were visualized to discriminate potential distinct strains. Finally, distinct yogurt isolates were identified by 16S rRNA gene sequencing and details for 16S rRNA gene PCR and sequencing were given elsewhere.[15] The phylogenetic relationship of the isolates was tested by MEGA7 program, and the evolutionary history was inferred using the Neighbour-Joining methodology.[17]
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.
Occupational exposure to anaerobic bacteria in a waste sorting plant
Published in Journal of the Air & Waste Management Association, 2021
Marcin Cyprowski, Anna Ławniczek-Wałczyk, Agata Stobnicka-Kupiec, Rafał L. Górny
Isolated DNA was used as a template for PCR reaction with primer pair specific to Clostridium genus: Chis150f (5ʹ-AAAGGRAGATTAATACCGCATAA-3ʹ) and ClostIr (5ʹ-TTCTTCCTAATCTCTACGCA-3ʹ) (Hung et al. 2008), which allow amplification of the gene fragment encoding 16S rRNA. The reaction mixture (20 μl) contained 2 μl of 10× reaction buffer with MgCl2, 0.5 U of RUN-HS Taq Polymerase (A&A Biotechnology), 250 µM of each deoxynucleotide (dNTP), 50 pmol of each primer and 0.5 μl of template DNA. Amplification included 35 cycles preceded by initial denaturation (95°C, 5 min). Each cycle included denaturation (95°C, 15 sec), annealing (58°C, 60 sec) and elongation (72°C, 60 sec) steps. The reaction ended with a final elongation (72°C, 5 min). The size of PCR product and the specificity of the primers were checked using electrophoretic analysis in 1.5% agarose gel (Certified™ Molecular Biology Agarose, BioRad, USA) and comparing the product size to DNA fragment marker (GeneRuler 1kb DNA Ladder, Thermo Scientific, USA).