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PCR-RFLP
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
Genomic DNA (200 to 300 ng) is amplified by the PCR procedure with 2.5 U of the Taq polymerase. The reaction mixture contains genomic DNA; 50 mM KC1; 2.5 mM MgQ2; 10 mM Tris-HCl, pH 8.4; 0.01% gelatin; 0.02% NP-40; and 200 μM each of dATP, dCTP, dTTP, and dGTP; 1 mM of each of the 5′ and 3′ primers in a total volume of 100 or 50 μl is covered with 50 μl of mineral oil and subjected to 30 c of 1 min for denaturing, 1 min for annealing, and 2 min for extension by automated PCR thermal sequencer or cycler. PCR cycling temperatures, depending on the primers used, are indicated in Table 2.
Molecular Genetics and Diagnostic Testing
Published in Merlin G. Butler, F. John Meaney, Genetics of Developmental Disabilities, 2019
The PCR process increases the number of copies of a specific DNA region by 100 million times in 3–4 hr. Therefore, the technique is capable of amplifying a minute amount of DNA to produce a large amount in a short period of time. The Taq polymerase is highly reliable, so that the sequence of the PCR product is an exact copy of the target region and may be analyzed for specific mutations or for polymorphic alleles for linkage.
Molecular biology
Published in Maxine Lintern, Laboratory Skills for Science and Medicine, 2018
PCR consists of three basic steps. The first is a heating step, usually up to about 95°C for about a minute, which denatures the target DNA into single strands. The temperature is then cooled to around 50-70°C, and specially designed primers (short sections of DNA which are complementary to the target) anneal to the DNA and provide a starting point for the Taq polymerase. The final step is DNA synthesis (72°C), where the Taq generates new DNA strands using the dNTPs (deoxyribonucleotide triphosphates) provided in the mix, starting at the primers and following the original template strand (seeFigure 10.3).
Resveratrol Blunts Mitochondrial Loss in Slow and Mixed Skeletal Muscle Phenotypes of Non-Human Primates following a Long-Term High Fat/Sugar Diet
Published in Journal of Dietary Supplements, 2023
Jon-Philippe K. Hyatt, Rafael de Cabo, Julie A. Mattison
Total DNA was extracted using a modified protocol detailed by Strauss (35). Approximately 45–60 mg of 15 µm-thick cross-sectioned muscle samples were collected and digested at 40 °C for 18 h in buffer containing 1 M NaCl, 0.1 M Tris-HCl, 0.1 M EDTA, 10% SDS, and fresh 0.1 mg/mL proteinase K. DNA was then extracted using standard phenol:chloroform:isoamyl alcohol methods (35) and resuspended in 1X Tris-EDTA buffer (pH 8.0). Semi-quantitative end-point PCR was performed using primers generated against the rhesus macaque nuclear and mitochondrial targets including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cytochrome c oxidase subunit II (COX2) and cytochrome b (CYTB) are shown in Table 1. Samples were prepared using 2X master mix containing optimized concentrations of Taq polymerase, dNTPs, MgCl2 (Promega, Madison, WI); 100 ng of DNA per reaction per sample was used. The cycling conditions consisted of one cycle at 94 °C for 5 min, 27 cycles at 94 °C for 30 sec, 27 cycles at 58 °C for 45 sec, 27 cycles at 72 °C for 45 sec, and a final extension at 72 °C for 5 min (Benchmark, Edison, NJ). Amplicons were separated in a 0.8% agarose gel containing 1X GelGreen (Biotium, Freemont, CA) at 45 V for 60 min, photographed and quantified using densitometry. The mitochondrial-to-nuclear gene ratio was then determined and statistically compared.
Valsartan-mediated chronotherapy in spontaneously hypertensive rats via targeting clock gene expression in vascular smooth muscle cells
Published in Archives of Physiology and Biochemistry, 2022
Jiajie Luan, Kui Yang, Yanyun Ding, Xiaotong Zhang, Yaqin Wang, Haiju Cui, Deixi Zhou, Lu Chen, Zhangqing Ma, Wusan Wang, Wen Zhang, Xiaoyun Liu
Total RNA was isolated from approximately homogenised organic tissue (100 mg) via Trizol Reagent (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA concentrations and purities were detected by a ND5000 Ultraviolet Visible Spectrophotometer (BioTeke Corp., Beijing, China). Approximately 1 μg of total RNA was reverse transcribed to cDNA via a PrimeScript RT reagent kit with gDNA Eraser (TAKARA, Japan), according to the manufacturer’s instructions. The obtained cDNA reaction (10 μl) was diluted 1:10 in DEPC-treated water to reduce inhibition of Taq polymerase. Diluted cDNA (2 μl) was added into a 10-μl PCR reaction containing specific primers (3 μl) for clock or housekeeping genes (Table 1) and commercial SYBR Green (5 μl), according to the manufacturer’s instructions. All primers were purchased from Generary Biotech Co., Ltd. (Shanghai, China).
True or false: what are the factors that influence COVID-19 diagnosis by RT-qPCR?
Published in Expert Review of Molecular Diagnostics, 2022
Luina Benevides Lima, Felipe Pantoja Mesquita, Lais Lacerda Brasil de Oliveira, Francisca Andréa da Silva Oliveira, Maria Elisabete Amaral de Moraes, Pedro F. N. Souza, Raquel Carvalho Montenegro
Overall, PCR reactions are applied to samples composed of DNA, allowing direct amplification by Taq polymerase activity and detection by the machine. However, to detect RNA viruses, like SARS-CoV-2, the process is a bit different (Figure 2). In this context, a previous step for viral mRNA conversion to DNA is required. Then, the RT-qPCR detection for RNA viruses occurs in two steps: 1) a reverse transcription reaction to produced complementary DNA (cDNA) using copies of mRNA as primer catalyzed by an RNA-dependent DNA polymerase (reverse-transcriptase) Taq polymerase is applied to amplify the specific segment of genome which provide result about virus presence (Figure 2) [20]. Most RT-qPCR tests for SARS-CoV-2 are quantitative by using fluorescence measurements that are sometimes referred to as RT-qPCR. Briefly, cDNA polymerizes with a probe targeted with both fluorescent and quencher labels. After polymerization into double-stranded DNA (ds-DNA), the quencher and fluorescent probes are separated and light emission from the fluorophore is observed upon light excitation [20].