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Salmonella typhimurium
Published in Yuli Rahmawati, Peter Charles Taylor, Empowering Science and Mathematics for Global Competitiveness, 2019
M. Nurjayadi, U.R. Efrianti, N. Azizah, E. Julio, L. Nastassya, V. Saamia
S. typhimurium infection through food contamination is a major public health threat even in developed countries, and is better known as a foodborne disease (Won and Lee, 2017). The fljB gene is an abundant protein-coding on the surface of the bacterial flagellum (Lim et al., 2003). This gene can be used as a primer in the Polymerase Chain Reaction (PCR) method to multiply the number of DNA molecules on a particular target, by synthesizing new DNA molecules that complement with the DNA molecule target. PCR is a repetitive reaction which includes template denaturation, annealing (pairing) the pair of primers on a single strand of DNA target, and extension (elongation) which can obtain DNA amplification.
Biomems
Published in Simona Badilescu, Muthukumaran Packirisamy, BioMEMS, 2016
Simona Badilescu, Muthukumaran Packirisamy
DNA amplification is the production of multiple copies of a sequence of DNA by using a process called polymerase chain reaction (PCR). PCR is the in vitro multiplication of defined strands of DNA with the use of enzymatic polymerase molecules. The technique allows a small amount of DNA to be amplified exponentially in order to produce enough DNA to be adequately tested. Since its first introduction in 1985, PCR-based techniques have become the most important part of DNA diagnostic laboratories because nearly all molecular biological operations need PCR to enrich the nucleic acid samples. At present, PCR is a widely utilized scientific tool in DNA sequencing, geno-typing, and new drug discovery. Due to its high specificity, PCR will play a major role in the future in the success of individualized medicine and point-of-care (POC) diagnostics. The discovery of this technology earned its inventor, K. B. Mullis, a Nobel Prize in Chemistry in 1993. While qualitative PCR has become a well-established technology, the quantification of specific target DNA sequences has been a difficult task. The concentration of a DNA species is nearly doubled in every PCR process, involving stepping through three different temperatures. In this way, the DNA concentration can be multiplied more than a million-fold by twenty to thirty cycles of temperature, and the DNA/RNA inside the PCR mixture will be rich enough for further analysis. Conventional PCR devices are suitable for amplifying DNA, but they are sizable devices and require long cycling times. Recently, miniaturized PCR devices have attracted great interest because of their advantages over conventional PCR devices, such as portability, low cost, fast thermal cycling speed, reduced reagents/sample consumption, low power consumption, and low cost of fabrication. Portability is of great advantage for environmental screening, as well as in the medical sector. In addition, smaller sample volumes have less heat capacity, allowing rapid temperature change. PCR microfluidic technologies have facilitated DNA amplification with improved performances.
Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Eva Agustriana, Isa Nuryana, Fina Amreta Laksmi, Kartika Sari Dewi, Hans Wijaya, Nanik Rahmani, Danu Risqi Yudiargo, Astadewi Ismadara, Moch Irfan Hadi, Awan Purnawan, Apridah Cameliawati Djohan
The large-fragment Bst DNA polymerase is the most widely used in biomedical applications and is commercially produced by industrial companies. In the diagnostic test, the enzyme has been applied for the Loop-mediated isothermal amplification (LAMP) method. In contrast with PCR-based DNA amplification, LAMP does not require a denaturation process for separating two DNA strands. The method has been developed by imitating the process of DNA replication in vivo. The physical process by heating for double-stranded DNA separation can be omitted and replaced with the enzymatic process by helicase[24]. In living organisms, a DNA helicase is functionated to disclose the double-stranded DNA into single strands, and subsequently, a DNA polymerase is involved in the synthesis and polymerization of new DNA strands during DNA replication[25]. In other words, the combination of these enzymes with a lack of 5′→3′ exonuclease activity has been included in the large fragment Bst DNA polymerase, therefore, DNA amplification can be performed under an isothermal condition in a simple way using one temperature from the scratch to the end of the reaction instead of thermocycling reaction.