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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.
Laboratory analyses of cyanobacteria and water chemistry
Published in Ingrid Chorus, Martin Welker, Toxic Cyanobacteria in Water, 2021
Judit Padisák, Ingrid Chorus, Martin Welker, Blahoslav Maršálek, Rainer Kurmayer
PCR is the technique that allows creating multiple copies of specific gene fragments through amplification by DNA polymerases. The most critical step for the reliable detection of toxigenic cyanobacteria is the selection of appropriate oligonucleotides (primers) which are used as molecular probes. Besides standard laboratory equipment, the instrumentation comprises a PCR cycling machine, a gel electrophoresis chamber and a gel documentation device. For detailed information on how to perform PCR, see the widely available laboratory manual revised by Sambrook and Russell (2001).
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).
Microbiology in Water-Miscible Metalworking Fluids
Published in Tribology Transactions, 2020
Frederick J. Passman, Peter Küenzi
Amplification and subsequent detection of nucleic sequences (DNA, mRNA, rRNA) are often done by application of PCR, a molecular technique that uses the ability of DNA polymerase to synthesize new strands of DNA complementary to the offered template strand. This technique can be used for microbial enumeration as well as for specification. Originally developed in the 1970s and 1980s, it has experienced many alterations and modifications since (194). Frequently, the invariable 16S rRNA gene (18S rRNA in eukaryotes) is targeted for identification and enumeration of OTUs. However, the technique is also useful for the detection of specific genes or the determination of transcriptional activity (enzyme-mediated process by which DNA gene codes are copied into messenger RNA) and is particularly useful for the detection of slow-growing microorganisms such as mycobacteria (195) in MWFs.
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
DNA polymerase is an important enzyme used for various applications and processes including routine PCR, real-time PCR, error-prone PCR, site-directed mutagenesis, DNA labeling and sequencing.[12,16] In addition, DNA polymerases are used for applications in synthetic biology and DNA-based computing.[17,18] Here we report gene expression optimization and high-recovery affinity purification of Pca-Pol. This enzyme has been shown to amplify DNA fragments up to 7.5 kb and used for DNA labeling application as well.[14]