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Application of Molecular Tools and Biosensors for Monitoring Water Microbiota
Published in Maulin P. Shah, Wastewater Treatment, 2022
PCR is one of the most commonly used molecular techniques for pathogen detection. It works on the principle of exponential amplification of a specific DNA sequence of the target organism. PCR is a cyclic process that includes three major steps: denaturation, annealing, and extension (36). PCR is a powerful technique that can be efficiently used for detecting a low quantity of target DNA in the environment sample due to its high sensitivity and specificity. Various modifications in the conventional PCR have led to the development of different variants of PCRs such as multiplex PCR (mPCR), reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qPCR) for pathogen detection.
Forensic DNA Profiling and Molecular Identification of Infectious Pathogens
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
D. E. Agbonlahor, M. Y. Tatfeng, Ifeoma B. Enweani-Nwokelo, Ifeoma M. Ezeonu, E. A. Brisibe, Francisca Nwaokorie, Nwadiuto (Diuto) Esiobu, A. O. Eremwanarue, F. E. Oviasogie, Benjamin Ewa Ubi, G. S. George
PCR technology has been applied in various fields of biotechnology including forensics (DNA profiling), identification of infectious diseases, cloning, DNA parentage testing, diagnosis of hereditary diseases, and for the analysis of different environmental samples (Butler, 2015). Repetitive DNA regions located outside the coding regions are used in forensics for DNA profiling. These regions vary from one individual to the other and can be used for identification of one person as well as a group of people, such as a group of family members. PCR can make copies of specific nucleotide sequences from a genome or degraded DNA. PCR uses a small amount of template DNA, two primers that bracket the sequence of interest, nucleotides, and thermostable DNA polymerase to amplify a specific region of DNA, thus creating a large amount of DNA from a very small sample.
Cloning of genes for protein expression
Published in Raimund J. Ober, E. Sally Ward, Jerry Chao, Quantitative Bioimaging, 2020
Raimund J. Ober, E. Sally Ward, Jerry Chao
An approach that is an alternative to the addition of restriction sites and restriction enzyme digestion is called TA cloning (Fig. 7.7). This is based on the activity of Taq polymerase (the DNA polymerase used in the PCR; see Section 7.2.2) to add an adenine that is not encoded by the template to the 3′ end of the DNA strand that is synthesized. Consequently, the PCR product has 3′ overhangs of adenine bases. These products can be ligated into vectors that have complementary, single-base thymine overhangs.
Pathogen contamination of groundwater systems and health risks
Published in Critical Reviews in Environmental Science and Technology, 2023
Yiran Dong, Zhou Jiang, Yidan Hu, Yongguang Jiang, Lei Tong, Ying Yu, Jianmei Cheng, Yu He, Jianbo Shi, Yanxin Wang
Polymerase chain reaction (PCR) provides a feasible diagnostic approach for pathogen detection. The PCR-based approaches significantly improve the efficiency of pathogen detection, especially for those difficult to be cultured (Bae et al., 2022). With designed primers, Taq polymerase, and temperature cycling created by a thermocycler, PCR can sensitively amplify the targeted DNA fragments from the genomes of potential pathogens (Garibyan & Avashia, 2013). In the past decade, significant advances in PCR-based approaches have been achieved. For example, multiplexed PCR (mPCR) can simultaneously detect multiple pathogens in a single reaction with a mixture of different pairs of primers (He et al., 2022). The limitations for PCR-based methods include inhibited efficiency of DNA extraction or defected activity of the polymerase by a wide range of contaminants commonly found in environmental samples. Meanwhile, false positive results due to contamination from extraneous naked nucleic acids can lead to misleading information for pathogen detection. In addition, as molecular detection with PCR requires a priori knowledge of the suspected microorganisms, it offers little information for the emerging or novel pathogens and the overall microbial community dynamics.
Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application
Published in Preparative Biochemistry & Biotechnology, 2023
Kashif Maseh, Syed Farhat Ali, Shazeel Ahmad, Naeem Rashid
Polymerase chain reaction (PCR) is a valuable technique used in genetic engineering, diagnostics and forensics. Over time, many technological advancements have been made in this field.[6] Various PCR-based methods are used for diagnosis of diseases and infections.[7–9] PCR involves heating at high temperatures, so a thermostable DNA polymerase is required for this process.[4] Hyperthermophilic archaea are particularly important in this regard, as their DNA polymerases are stable at high temperatures.[5] Archaea are known to have DNA polymerases belonging to B-family (PolB) and D-family (PolD).[10] Based on sequence and phylogenetic analyses, archaeal PolBs have been characterized into various groups, some of which have close relationship with their eukaryotic counterparts.[11] Various B-family archaeal DNA polymerases have been characterized and some are commercially available as well.[12]
Bacterial community in commercial airliner cabins in China
Published in International Journal of Environmental Health Research, 2020
Mingxin Liu, Junjie Liu, Jianlin Ren, Lumeng Liu, Ruiqing Chen, Yanju Li
Polymerase chain reaction technology (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as special DNA replication in vitro. The most prominent feature of PCR is that it can greatly increase trace amounts of DNA. PCR is the use of DNA denaturation, which will become single-stranded at high temperature (95°C) in vitro. When the temperature is low (usually around 60°C), the primers are combined with single-stranded base pairing. When adjusting the temperature to the DNA polymerase optimal reaction temperature (about 72°C), DNA polymerase along the direction of phosphoric acid to five carbon sugar synthesizes a complementary strand. With the characteristics of strong specificity, high sensitivity, simplicity, speed, and low purity requirements, PCR technology has been widely used in 16s rRNA identification.