DNA Analysis
Steven H. Y. Wong, Iraving Sunshine in Handbook of Analytical Therapeutic Drug Monitoring and Toxicology, 2017
The PCR amplification process involves repeated cycles of three steps: 1) denaturation—splitting of the double-stranded DNA into single-stranded DNA by literally melting the strands apart; 2) annealing—the binding of two small single-stranded pieces of DNA, primers, to the opposite strands bracketing the area of interest; and 3) extension—replication of the strands being created from the extension of the two primers by a DNA polymerases48 The result is a doubling of target DNA for each three-step cycle. The newly synthesized DNA strand serves as a template for the next cycle. The Taq polymerase from the Thermus aquaticus bacteria which grow in thermal geysers permits the use of high temperatures without significant denaturation of the enzyme. This enzyme has made PCR amenable to automation. The different steps proceed at different temperatures with the same reaction mixture; and therefore, PCR can be carried out in an instrument called a “thermal cycler”.
The science of biotechnology
Ronald P. Evens in Biotechnology, 2020
PCR is a critical core process in biotechnology that permits substantial expansion of the amount of genetic material (DNA, genes), starting from minute amounts. As discussed in Chapter 2, first, the process involves denaturing DNA with high heat (90°C), that is, unraveling the DNA double helix so that the genetic code (sequence) can be read and possibly duplicated. Second, a leader sequence for DNA is used to bind to the target DNA sequence and initiates reading of the genetic code at a specific point in DNA. Both helices (strands) of DNA can be read, that is, duplication of the target DNA sequence. Third, the heat-stable enzyme from the bacteria Thermus aquaticus, a DNA polymerase, catalyzes the reading of the genetic code with incorporation of the four nucleotides and extension of the replicated DNA sequence. The four nucleic acids are provided as sources for DNA duplication (adenine, thymine, guanine, cytosine). By sequential repetition of these three steps, the genetic material is magnified; for example, 20 replications of the three steps yield a million-fold increase in the DNA material.
Genetics and deoxyribonucleic acid-based technology in clinical biochemistry
Martin Andrew Crook in Clinical Biochemistry & Metabolic Medicine, 2013
Taq polymerase is a heat-stable DNA polymerase derived from the organism Thermus aquaticus. Polymerase chain reaction (PCR) can amplify DNA up to a million-fold. It can be used in the diagnosis of known mutations of genomic DNA using an amplification refractory mutation system (ARMS). DNA cannot undergo PCR amplification if an oligonucleotide primer contains a 3′ nucleotide mismatch. In site-directed mutagenesis, a mutation or polymorphism destroys or creates a restriction endonuclease cutting site, which can be identified by PCR followed by endonuclease digestion (Fig. 28.3). The ARMS allows diagnosis of single nucleotide mutations, where specific priming of PCR allows amplification to take place only if the mutation is present. Gene sequencing can also be used in some cases to diagnose certain mutations.
Specific FSTL1 polymorphism may determine the risk of cardiomyopathy in patients with acromegaly
Published in Acta Cardiologica, 2022
Suleyman Nahit Sendur, Tuncay Hazirolan, Busra Aydin, Incilay Lay, Mehmet Alikasifoglu, Tomris Erbas
Five to 10 mL peripheral venous blood samples were collected from acromegalic patients. DNA was isolated via ammonium acetate salt precipitation. For the measurement of genomic DNA concentration and purity, a spectrophotometer was utilised. To assess the quantity and purity of DNA, 260 and 280 nm wavelengths values were used. A260/280 and A260/230 ratios were identified to evaluate DNA cleansing. The regions containing a polymorphism (rs1259293), which was previously identified in the literature, were amplified using the polymerase chain reaction (PCR) method [26]. One pair of primers were designed using Primer3® (http://bioinfo.ut.ee/primer3-0.4.0/) for amplification. Taq DNA polymerase enzyme (Thermo Fisher Scientific®, USA) was used for PCR (Thermus aquaticus). The PCR was completed under the amplification conditions determined on the Veriti thermal cycler (Thermo Fisher Scientific®, USA) and the products were checked on an agarose gel.
Related Knowledge Centers
- Bacteria
- Molecular Biology
- Oligopeptide
- Polymerase Chain Reaction
- Protease
- Taq Polymerase
- Amino Acid
- Deinococcota
- Species
- Chemotroph