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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
PCR products, which are double-stranded DNA (dsDNA), are invisible to the naked eye and thus require specific methods to enable visualisation. There are a variety of ways of visualising PCR fragments, including DNA fragment separation and visualisation, in which DNA fragments are separated by size on an agarose gel followed by ethidium bromide binding and UV light visualisation; restriction enzyme digestion and visualisation, in which PCR products are cut with a restriction enzyme (that recognise a specific sequence) and then separated on an agarose gel followed by ethidium bromide-UV light visualisation; fluorescent labelling of alleles, in which fluorophores (fluorescent primers or hybridisation probes) are added to the PCR reaction and the thermal cycler is designed to analyse the colour directly at the end of the PCR reaction, with different DNA variants producing different colours that correspond to a given genotype; and DNA sequencing, in which the PCR fragments are directly sequenced using a DNA sequencer that reveals all of the bases within the whole DNA/PCR product.
The Precision Medicine Approach in Oncology
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Measurements of genetic variability can be used to establish a link between the variations and disease, and to predict a patient’s therapeutic response to a drug along with potential toxicological outcomes. After completion of the Human Genome Project (HGP) between 2000 and 2003, the next important step was the discovery of new nonredundant genetic markers known as Single-Nucleotide Polymorphisms or “SNPs”. This involved DNA isolation followed by nucleic acid amplification and product detection. Originally, this was a laborious process involving Southern Blots and manual di-deoxy sequencing. The first technological revolution was based on the polymerase chain reaction (PCR), and included assays such as PCR Restriction Length Fragment Polymorphism (PCR-RLFP), PCR-ligase Detection, PCR Fluorescent Resonance Energy Transfer (PCR-FRET), and the nonamplified FRET assay (Invader Assay). The next improvement resulted from automated sequencing instruments (i.e., DNA Sequencers), which allowed high-throughput nucleotide sequencing and pyrosequencing. Finally, a revolution in SNP analysis resulted from the development of SNP Arrays and accompanying bioinformatics software that enabled large-scale linkages to be identified along with association and copy number studies in a low-cost, high-throughput fashion. Based on these developments, a large range of commercial instruments is now available that allow simultaneous measurement of thousands of SNPs across numerous samples using standard protocols, reagents, and data analysis systems.
Diagnosis: Nanosensors in Diagnosis and Medical Monitoring
Published in Harry F. Tibbals, Medical Nanotechnology and Nanomedicine, 2017
Ion Torrent Systems is another new company that is developing disruptive massively parallel technology for high-throughput, low-cost, rapid DNA sequencing. Pacific Biosciences of California is developing a DNA sequencer chip called SMRT (single-molecule, real-time) with the goal of sequencing an entire human genome in 15 min by 2013, using a shotgun process with libraries of very long strings.
Gene expression and six single nucleotide polymorphisms of interleukin-6 in rheumatoid arthritis: A case-control study in Iraqi patients
Published in Alexandria Journal of Medicine, 2018
Ali H. Ad'hiah, Aseel S. Mahmood, Abdul-Kareem A. Al-kazaz, Khadier K. Mayouf
The Genomic DNA was extracted from EDTA blood using ReliaPrepTM Blood gDNA Miniprep System (Promega Corporation, USA), and after assessing purity and concentration, it was subjected to PCR amplification. Forward (5′-GGAGTCACACACTCCACCT-3′) and reverse (5′-CTGATTGGAAACCTTATTAAG-3′) primers of IL6 were adopted from a previous study.12 The PCR reaction was performed in a final volume of 25 µl, which included 12.5 µl GoTaq green Master mix, 0.75 μl of each primer (10 μM), 2 µl DNA sample (50 ng) and 9 µl nuclease-free distilled water. The PCR conditions were initial denaturation at 95 °C for 5 min (one cycle), followed by 35 cycles of denaturation at 95 °C (30 s), annealing at 60 °C (30 s) and extension at 72 °C (30 s), followed by a final extension at 72 °C for 7 min. The amplified PCR fragments were subjected to Sanger’s sequencing using ABI3730XL automated DNA sequencer (Macrogen Corporation – Korea). The IL6 gene SNPs (rs1800796, rs7802307, rs7802308, rs36215814, rs184229712 and rs867254801) and their genotypes were revealed by the Geneious software version 10.2.2 after alignment with reference sequences in the Gene Bank.
The impact of FLT3 mutations on treatment response and survival in Chinese de novo AML patients
Published in Hematology, 2018
Qiao-Cheng Qiu, Chao Wang, Xie-Bing Bao, Jing Yang, Hong-Jie Shen, Zi-Xuan Ding, Hong Liu, Jun He, Hong Yao, Su-Ning Chen, Zheng Li, Sheng-Li Xue, Song-Bai Liu
All samples investigated in this study were obtained at the time of diagnosis. Genomic DNA was extracted from BM samples according to the manufacture’s protocols (QIAGEN AllPrep DNA/RNA mini kit; QIAGEN). As the location of FLT3-ITD is restricted to exons 14 and 15, polymerase chain reaction (PCR) was performed using the method described by Meshinchi et al. [23]. In brief, 100 ng of DNA was amplified in a volume of 50 µl containing 50 mM KCl, 10 mM Tris–HCl, pH 8.3, 1.5 mM MgCl2, 0.001% (wt/vol) gelatin, 200 µM dNTPs, 0.5 µM of each oligonucleotide (FLT3-ITDF: 5′-GCAATTTAGGTATGAAAGCCAGC-3′ and FLT3-ITDR: 5′-CTTTCAGCATTTTGACGGCAACC-3′), and 1 unit Taq DNA polymerase (QIAGEN, Hilden, Germany). The PCR consisted of an initial incubation step at 95°C for 180 seconds, followed by 35 cycles at 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 90 seconds, and a final elongation step at 72°C for 10 minutes. PCR products were analyzed on standard 3% agarose gels. A portion of each PCR products were sequenced using an ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, U.S.A.). PCR for detection of FLT3-TKD mutations was performed using the same method as above, and the oligonucleotides are: 5′-CCACATCAACGCTATGAGGTAA-3′ (FLT3-TKDF) and 5′-CAGCCCAGTAAAGATAAGAGGC-3′ (FLT3-TKDR).
Association of TP73-AS1 gene polymorphisms with the risk and survival of gastric cancer in a Chinese Han Population
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Wangwang Chen, Jian Xiao, Liang Shi, Linling Lin, Mingkun Jiang, Yugang Ge, Zengliang Li, Hao Fan, Li Yang, Zekuan Xu
We obtained 5 ml venous blood from each subject and extracted DNA by a standardized kit following the manufacturer’s protocols as presented previously [21]. The selected SNPs (rs1181865, rs9800, rs3737589, rs2298222, rs7515164) were genotyped by the polymerase chain reaction-ligase detection reaction (PCR-LDR) sequencing method. The length of DNA fragments and the corresponding primer sequences can be seen in Supplemental Table S1. PCR primer sequences were designed by using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast). The multiplex PCR was carried out in the reaction volume of 15 µl containing 2 µl DNA template, 7.5 µl 2× HotTaq PCR Reaction Mix, 2 µl mixed primers, and 3.5 µl ddH2O. The reaction conditions were as follows: pre-denaturation at 94 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, at last extension at 72 °C for 3 min. LDR was performed in the reaction volume of 10 µl containing 3 µl multiplex PCR product, 1 µl 10 × Taq DNA ligase buffer, 0.125 µl 40 U/μl Taq DNA ligase, and 3 µl 1 pmol probes (0.2 µl each of probe), supplemented with ddH2O to reach the total volume of 10 µl. The corresponding probe sequences can be seen in Supplemental Table S2. Probe sequences are fixed sequences before and after the SNP sites in TP73-AS1. The reaction conditions were 30 cycles of 94 °C for 30 s and 56 °C for 3 min. The LDR products were analyzed on an ABI 3730XL DNA sequencer. This work was carried out by the Shanghai Generay Biotech Co. Ltd (http://www.generay.com.cn). In addition, 10% of the samples were selected randomly to repeated assays for quality control, and the final concordance rate was 100%.