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Markers of Sensitivity and Resistance to EGFR Inhibitors in Colorectal Cancer
Published in Sherry X. Yang, Janet E. Dancey, Handbook of Therapeutic Biomarkers in Cancer, 2021
Jose G. Monzon, Janet E. Dancey
To detect these mutations, studies have typically employed direct sequencing of exons 9 and 20 or mass spectrometry [90, 119–121]. The latter involves PCR amplification of the region containing the mutation of interest, an extension reaction to generate allele-specific DNA products, and chip-based mass spectrometry for separation and analysis of the DNA. A single base extension reaction was performed using extension primers that hybridize immediately adjacent to the mutation. The extension reaction generates diagnostic products that, based on their unique mass values, allow discrimination between two alleles. This method is very sensitive, but only known mutations can be tested and it sometimes requires direct sequencing for verification.
Laboratory Molecular Methodologies to Analyze DNA Methylation
Published in Cristina Camprubí, Joan Blanco, Epigenetics and Assisted Reproduction, 2018
Each individual bead is conjugated to an oligonucleotide comprising a 23 bp address to allow identification of their physical location on the BeadChip and a 50 bp probe that is designed complementary to the bisulfite-converted DNA with a CpG site at the 3′ end of the probe. After hybridization to bisulfite converted DNA, single-base extension of the probe incorporates a fluorescently labeled ddNTP at the 3′ CpG site to allow “genotyping” of the C/T bases that result from bisulfite conversion.
Detection Techniques for Single Nucleotide Polymorphisms
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
W. Mathias Howell, Johan Stenberg, Chatarina Larsson, Mats Nilsson, Ulf Landegren
The GenomeLab™ SNPstream genotyping system of Beckman Coulter (Fullerton, California, USA, www.beckman.com/) is based on the SNP-IT™ assay99,100 originally marketed by Orchid Cellmark (Princeton, New Jersey, http://www.orchid.com/). Essentially, the method entails single-base extension (SBE) or “minisequencing” followed by fluorescence detection on tag arrays. The assay procedure starts by multiplex PCR amplification of up to 12 different SNP loci. After purification, locus-specific primers are added and annealed adjacent to the SNP position. Each primer contains a unique tag sequence. A cyclic, allele-specific extension reaction ensues in which fluorescence-labeled ddNTPs are added to the extension primers.
Associations of the KiSS-1 and GPR54 genetic polymorphism with polycystic ovary syndrome in Yunnan, China
Published in Gynecological Endocrinology, 2022
Ting Zhao, Qiong Zhang, Xiao Xiao, Xinghua Tao, Meixiu Gao, Wenli He, Xiaomei Wu, Tao Yuan
Genomic DNA was extracted from 2 ml of peripheral blood leukocytes following the manufacturer’s protocol (QIAGEN, USA). We determined the purity and concentration of DNA using a Nanodrop spectrophotometer. We searched the target sequences of KiSS-1 and GPR54 single-nucleotide polymorphism (SNP) sites on the NCBI website (https://www.ncbi.nlm.nih.gov/), and then designed the peripheral amplification primers. We used Primer Premier 5 for designing single-base extension primers for all sites. The polymerase chain reaction (PCR) amplification conditions consisted of an initial denaturation step at 95 °C for 15 min, followed by 34 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. We confirmed the purity and size of the PCR product, and then proceeded with nucleotide sequencing using an ABI Big Dye Terminator protocol on an ABI 3730XL Avant Genetic Analyzer. Table 1 lists the gene polymorphisms in the participants.
KANK1 regulates paclitaxel resistance in lung adenocarcinoma A549 cells
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2020
Junyi Pu, Jianfeng Shen, Zihua Zhong, Ma Yanling, Jie Gao
A total of 500 ng DNA of each sample was used as input material for DNA sample preparation. Sulphite transformation was carried out using the Zymo EZ DNA Methylation Kit recommended by Illumina, following the manufacturer’s protocol. 0.1 N NaOH was added to the sample to denature the DNA. After neutralisation, genome-wide amplification reagent was added and the solution was incubated overnight at 37 °C. Isopropanol was added, and DNA fragments were precipitated by centrifugation at 4 °C, the precipitated DNA was air dried, and hybrid buffer reagent was added to re-dissolve the DNA precipitate. During the hybridisation process, the fragmented DNA was denatured and annealed to 50 bases attached to the bead of the chip at a specific site. The un-hybridised and non-specifically hybridised DNA was washed, and the captured DNA was used as a template for a single base extension reaction on the chip, adding fluorescent groups on the chip, in order to distinguish the methylation status of samples.
A genetic link between CXCR5 and IL2RA gene polymorphisms and susceptibility to multiple sclerosis
Published in Neurological Research, 2018
Zong-Li Xia, Qing-Mei Qin, Qing-Ying Zhao
Venous blood of 3 mL was collected from each subject in the morning to extract the genomic DNA using TiangenTM DNA extraction kit (TIANGEN Biotechnology Co. Ltd, Beijing, China), followed by storing at −80°C. Next, sequenom MassARRAY system was employed for genotyping of CXCR5 (rs3922). The primers and probes of rs3922(C > T) locus, as shown in Table 1 were synthetized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The polymerase chain reaction (PCR) system consisted of 0.1 U TaqDNA polymerases, 5-ng genomic DNA, 2.5 pmol PCR primers, and 2.5 mmol dNTPs. PCR reaction conditions were as follows: 45 cycles of 94°C for 15 min and 94°C for 20 s, annealing at 56°C for 30 s and extension at 72°C for 30 s. The remaining dNTP was removed using 0.3 U alkaline phosphatases. The process of single base extension consisted of 5.4 pmol extended random primers, 50 μmol dNTP/ddNTP compounds, and 0.5 U Thermosequenase DNA polymerase. The reaction conditions were as follows: 40 cycles of 94°C for 2 min, 94°C for 5 s, 50°C for 5 s and 72°C for 5 s.