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Altitude, temperature, circadian rhythms and exercise
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Henning Wackerhage, Kenneth A. Dyar, Martin Schönfelder
Living organisms populate extreme “thermal niches”, ranging from polar species such as ice bears to thermophilic bacteria such as Methanopyrus kandleri that can survive and reproduce in temperatures up to 122°C (56). For the polymerase chain reaction (PCR), a key molecular biology method (see Chapter 2), biologists are using the genetically heat-resistant polymerase of thermophilic Thermus aquaticus as the PCR reaction requires temperatures of nearly 100°C.
The science of biotechnology
Published in Ronald P. Evens, 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.
Molecular biology
Published in Maxine Lintern, Laboratory Skills for Science and Medicine, 2018
The basic principle of the method exploits the fact that DNA is double-stranded, and if you denature the helix using high temperatures, you can make another complementary strand using DNA polymerase and a few loose bases, effectively doubling the amount of DNA you have. If you do that again and again, you can see how you can make lots of copies, all identical to the original strand. What makes this technique practical is the use of automation by engaging the services of the highly temperature-resistant DNA polymerase from Thermus aquaticus. This bacteria lives in hot springs, and is quite happy at temperatures around 100°C. Most other DNA polymerases only work at low temperatures, but DNA only denatures into two strands at high temperatures. Thus Taq polymerase can be used to make copies of DNA by cycling between high and low temperatures every few minutes, so in the space of a few hours you can amplify one copy of DNA up to millions! PCR machines costing a few thousand pounds are fundamentally programmable heating/cooling blocks that take your samples up and down to exact temperatures for an exact number of cycles, before cooling the amplified DNA ready for use. Many labs with high usage of PCR techniques will have banks of machines running 24 hours a day.
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.