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DNA Markers in Forensic and Diagnostic Science
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
M. Y. Tatfeng, D. E. Agbonlahor, Ifeoma B. Enweani-Nwokelo, Ifeoma M. Ezeonu, Francisca Nwaokorie, E. A. Brisibe, D. Esiobu
PCR is an important tool in genetic marker studies. It enables the amplification of a DNA segment of interest through an enzymatic and temperature controlled reaction. Three main steps are involved namely denaturation, annealing and extension. The components of a PCR reaction include the thermostable enzyme Taq which derives its name from the Bacillus thermoaquaticus from which it is harvested. This enzyme extends the newly synthesized strand by joining the nucleotides to each other. The deoxynucleotide triphosphates are the building blocks (deoxycytidine triphosphate, deoxyadenosine triphosphate, deoxyguanosine triphosphates and deoxythymidine triphosphates), the buffer maintains the reaction at physiological pH, the cations (magnesium chloride) enhance primer binding and Taq activity, the primers also known as oligonuleotides are short synthesized DNA fragment that bracket the DNA segment of interest during amplification, their length ranges between 18 and 30 base pairs with a recommended GC/AT ration of 50:50, and the template is the extracted genome housing the DNA fragment of interest. There are several types of PCRs: quantitative or Real-Time PCR, multiplex PCR, singleplex PCR, gradient PCR, reverse transcriptase PCR, touchdown PCR, etc.
Genes and Genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
PCR is a technique to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands of millions of copies of a particular DNA sequence. PCR was developed in 1983 by Kary Mullis. Now, it is a common and indispensable technique used in medical and biological research laboratories for a variety of applications such as DNA cloning for sequencing, DNA-based phylogeny, functional analysis of genes, diagnosis of hereditary diseases, identification of genetic fingerprints, and detection and diagnosis of infectious diseases. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers that are basically short DNA fragments containing sequences complementary to the target region along with a DNA polymerase are key components to enable selective and repeated amplification. As the PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can also be extensively modified to perform a wide array of genetic manipulations (Figure 2.16).
Genes and genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
PCR is a technique to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands of millions of copies of a particular DNA sequence. PCR was developed in 1983 by Kary Mullis. Now, it is a common and most indispensable technique used in medical and biological research laboratories for a variety of applications such as DNA cloning for sequencing, DNA-based phylogeny, functional analysis of genes, diagnosis of hereditary diseases, identification of genetic fingerprints, and detection and diagnosis of infectious diseases. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers that are basically short DNA fragments containing sequences complementary to the target region along with a DNA polymerase are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can also be extensively modified to perform a wide array of genetic manipulations (Figure 2.16).
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.