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Hereditary Cerebral Amyloid Angiopathy Associated with a Novel Amyloid Beta Precursor Protein Mutation
Published in Gilles Grateau, Robert A. Kyle, Martha Skinner, Amyloid and Amyloidosis, 2004
L. Obici, G. De Rosa, G. Palladini, S. Marciano, S. Donadei, E. Arbustini, L. Verga, M. Concardi, G. Ferrari, G. Merlini
Mutation analysis of exon 17 of the AβPP gene was performed by PCR amplification using the following, flanking intronic primers: 5’-ACC TCA TCC AAA TGT CCC CTG C-3’ and 5’-TCT CAT AGT CTT AAT TCC CAC-3’. Amplification conditions for AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA) were 95°C for 10 minutes for maximal enzyme activation, followed by 30 cycles at 95°C for 1 minute, 60°C for 1 minute, and 72°C for 1.5 minutes. PCR products were cleaned by gel extraction and both strands were sequenced with the Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and the products were analysed on an ABI PRISM 310 DNA sequencer (Applied Biosystems, Foster City, CA, USA).
Optimization of small RNA extraction and comparative study of NGS library preparation from low count sperm samples
Published in Systems Biology in Reproductive Medicine, 2021
Victoria Shtratnikova, Vladimir Naumov, Vitaly Bezuglov, Anna Zheludkevich, Luidmila Smigulina, Yury Dikov, Tatiana Denisova, Alexander Suvorov, J. Richard Pilsner, Russ Hauser, Stephen A. Krawetz, Oleg Sergeyev
Size selection of 147–160 nucleotides fragments was carried out using high-resolution gel containing 0.8% agarose (Bio-Rad, Hercules, CA, USA), 0.4% polygalactomannan (OOO ‘Izogel’, Puschino, Russian Federation) and 1.6% γ–polygalactomannan (OOO ‘Izogel’, Puschino, Russian Federation). The resolution of such gel is approximately equivalent to that of a 4% agarose gel. The dry mixture of reagents is previously suspended in ethanol (before dilution in buffer). After melting, hold at 65 °C for about 30 minutes until bubbles are removed. Due to the fragility of the gel, the subsequent pouring should take place strictly in the form where the electrophoresis will be performed. The thickness of the gel did not exceed 5-6 mm, otherwise the separation was not effective. Electrophoresis was performed at 120 V during 1 hour in the tris-acetate-EDTA (TAE) buffer. Elution from the gel was performed using the kit MinElute DNA Gel Extraction Kit (Qiagen, Netherlands).
Association study of CACNA1C polymorphisms with large artery atherosclerotic stroke in Chinese Han population
Published in Neurological Research, 2018
Chen Peng, Ying Ding, Xin Yi, Zhiqiang Dong, Limei Cao, Qiang Li, Haiyan Ren, Lin He, Daizhan Zhou, Xu Chen
The barcoded PCR products were analyzed using gel electrophoresis. And this step can ensure that the expected insert size of the barcoded PCR products was obtained. They were pooled together with equal volume and purified using AMPure XP beads. The targeted DNA fragment was selected and extracted using E-Gel Precast Agarose Electrophoresis (Thermofisher) and QIAquick Gel Extraction Kit. The library was quantified by Agilent Bioanalyzer, and then was sequenced in 2X150 bp paired-end model using the HiSeq X Ten platforms of Illumina Custom sequencing primers.
Identification of PITX3 mutations in individuals with various ocular developmental defects
Published in Ophthalmic Genetics, 2018
Celia Zazo Seco, Julie Plaisancié, Tatiana Lupasco, Caroline Michot, Jacmine Pechmeja, Julian Delanne, Edouard Cottereau, Carmen Ayuso, Marta Corton, Patrick Calvas, Nicola Ragge, Nicolas Chassaing
Primers for amplification and sequencing of exons and exon-intron boundaries of PITX3 (ENST00000370002) are shown in Table S2 in Supplementary Material. Amplification by Polymerase Chain Reaction (PCR) was performed on 25 ng of genomic DNA with Taq DNA polymerase (Life Technologies, Carlsbad, CA, USA). PCR fragments were purified with a gel extraction kit (Neo Biotech CliniSciences, Nanterre, France) in accordance with manufacturer’s protocol. Sequence analysis was performed with the 3500xL sequencer (Applied Biosystems, Foster City, CA, USA).