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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
The mastermix and the gDNA of each individual being studied are then combined in thin-walled PCR tubes or plates and inserted into a thermal cycler, which allows for rapid heating and cooling of the mixtures in the tubes. The ‘cycles’ of the thermal cycler are repeated cycles of denaturation, which causes the DNA double strand to separate; annealing, in which the sample is cooled, and the PCR primers bind to the gDNA region of interest; and extension, in which the sample is warmed again to a temperature at which Taq polymerase extends the DNA double strand at the point that the primers have annealed to the gDNA. The PCR cycles are repeated until a sufficient amount of gDNA is made, which is commonly over >25 cycles (see Figure 2.1).
HLA-DR Typing by Polymerase Chain Reaction Amplification with Sequence-Specific Primers (PCR-SSP)
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
The DR PCR-SSP typing system has been developed for fast second generation thermal cyclers. The main advantages of these new thermal cyclers are that they are rapid, very accurate, have a low thermal gradient across the heating block, small reaction volumes can be used and overlay with mineral oil is not necessary. Older thermal cyclers can be used, provided the thermal gradient across the heating block does not exceed 1°C. When using these thermal cyclers the reaction volumes must be increased to 25 μl, mineral oil must be used, thin-walled reaction tubes are preferred, the steps of the PCR cycle must in most cases be somewhat extended, and the annealing temperature might have to be increased 1°C to 2°C.
Quantitative Polymerase Chain Reaction for Companion Diagnostics and Precision Medicine Application
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Joel L. Ramirez, Johannes R. Kratz
After extraction of DNA, samples containing DNA are put into a thermal cycler along with primers, deoxyribonucleotides, buffer solution, and a heat-resistant DNA polymerase. By selecting the primers, the experimenter can effectively choose the genetic sequence of interest to be replicated. The thermal cycler allows for rapid cycling between hot and cold temperatures, which allows for repeated cycles of DNA replication. These cycles typically have three distinct stages. The first stage occurs at ~95°C and heating to this temperature causes separation of DNA’s double chain structure into two single strands of complimentary DNA. The second stage is characterized by decreasing the temperature to ~50°C, which allows for the binding of the DNA primers to the single strands of DNA. In the third stage, the temperature is increased to ~70°C, which allows for DNA polymerase to bind to the primer and single strands of DNA and undergo polymerization, creating two new pairs of double stranded DNA, identical to the original. This three-stage cycle gets repeated for 30–50 times.
Mutational analysis of minichromosome maintenance complex component (MCM) family genes in Chinese Han women with polycystic ovarian syndrome
Published in Gynecological Endocrinology, 2023
Jiangyan Zhou, Faying Liu, Lifeng Tian, Ming Yang, Jun Tan, Xianxian Liu, Peishuang Li, Jia Chen, Ge Chen, Lixian Xu, Lisha Peng, Qiongfang Wu, Yang Zou
The PCR was carried out in a final reaction volume of 25 μL containing ∼50 ng total DNA, 12 μL of Ampliqon master mix (TaKaRa, cat. no: RR320), 10 pmol of forward primer and 10 pmol of reverse primer. The PCR program involved an initial pre-denaturation step at 95 °C for 3 min, followed by 30 cycles, including a denaturation step at 95 °C for 30 s, a primer annealing step at different temperatures (52–60 °C, Supplemental Table 1) for 30 s, and an extension step at 72 °C for 30 s, final extension step at 72 °C for 7 min. Thermal Cycler 2720 (Applied Biosystems, Thermo Fisher Scientific, Inc.) was used for performing PCR. Briefly, 2 μL of PCR products were resolved by 2% agarose gel for evaluating the PCR products length. The PCR products were then purified and sequenced on an ABI Prism 3730 DNA sequencer (Applied Biosystems, Thermo Fisher Scientific, Inc.). The sequencing chromatograms were analyzed with DNAStar software. All the variants were confirmed by three independent PCR runs and sequenced in both forward and reverse strands. SIFT (http://sift.jcvi.org/) and PolyPhen2 (http://genetics.bwh.harvard.edu/), the online bioinformatic programs, were used to analyze the disease-causing potential of the identified variants.
Diagnostic approaches for dengue infection
Published in Expert Review of Molecular Diagnostics, 2023
Gaythri Thergarajan, Shamala Devi Sekaran
Nucleic acid sequence-based amplification (NASBA) is a one-step isothermal process for amplifying RNA that does not require the use of a thermal cycler. This technique has been proven to be successful in the detection of both viral and bacterial RNA in clinical samples. The NASBA assay involves the use of extracted nucleic acid and subsequently amplified at a constant temperature of 41°C. The reaction mixture consists of avian myeloblastosis reverse transcriptase (AMV-RT), T7 RNA polymerase, and RNase H with two short single-stranded DNA primers. The amplified product of RNA pools is then detected with agarose gel electrophoresis and electrochemiluminescence (ECL) signal count. NASBA has significant specificity and sensitivity, comparable with that of virus isolation, and may be particularly useful in field studies of dengue infection. Wu et al. reported that the NASBA assay had a sensitivity of 98.5% (66/67 clinical samples), and a specificity of 100% (21 normal human serum) when compared with the cell culture method using C6/36 cells [66]. The effectiveness of this method has also been described for detecting the dengue virus within mosquitoes [67,68]. It is considered more efficient than PCR in that it bypasses the use of a thermal cycler, which makes it both rapid (<1 day) and cost-effective.
Triptolide inhibits T-cell acute lymphoblastic leukaemia by affecting aberrant epigenetic events in the Wnt signalling pathway
Published in Journal of Chemotherapy, 2023
Yanna Ma, Ying Li, Mei Huang, Yuesheng Meng
DNA was extracted from T-ALL cell lines treated with TPL or not using QIAmp Tissue Kits. DNA was isolated according to the manufacturer’s protocol. MSP for aberrant gene promoter methylation was performed as previously described [16]. Treatment of 500 ng DNA with bisulphite for conversion of unmethylated cytosine to uracil (but not affecting methylated cytosine) was performed using an EZ DNA methylation kit according to the manufacturer’s protocol. The methylation status of SOX17, WIF1, SFRP5 and CDH1 was tested. Primers for the methylated (M-MSP) and unmethylated (U-MSP) promoters are from previous reports [17–19]. DNA from cells without TPL treatment was used as the control group, while cells treated with TPL were used as the experimental group. Then, PCR was performed in a thermal cycler with the following cycling conditions: 95 °C for 10 min, 37 cycles of 95 °C for 30 s, specific annealing temperature for 30 s, 72 °C for 30 s and a final extension of 7 min at 72 °C. PCR was performed using HS DNA Polymerase according to the manufacturer’s protocol. Then, PCR products were loaded onto 2% nondenaturing polyacrylamide gels, electrophoresed and visualized under ultraviolet light after staining with ethidium bromide. Each experiment was repeated three times.