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HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
DNA-restriction fragment length polymorphism (RFLP) typing is a well-established technique for routine identification of HLA-DR and -DQ allotypes. Methods and an interpretation scheme are presented which permit the identification of HLA-DR and -DQ allotypes using a single restriction endonuclease, TaqI. Resolution between problematic specificities may be rapidly achieved by simple polymerase chain reaction (PCR) -based supplementary tests.
The Genetics of Alzheimer Disease:
Published in Robert E. Becker, Ezio Giacobini, Alzheimer Disease, 2020
Molecular genetic techniques allow creation of innumerable DNA markers by cleaving DNA using restriction endonucleases. The resulting DNA fragments, also known as restriction fragment length polymorphisms (RFLPs), can then be used as markers in linkage analyses. Since RFLPs of different lengths from any chromosome can be produced, microdissection of the human genome is possible. More extensive discussion of such techniques are reviewed in another chapter of this book (Wizniewski, 1989).
Repeated DNA Sequences and Polyploidy in Cereal Crops
Published in S. K. Dutta, DNA Systematics, 2019
When repeated sequences do not contain the sites for a restriction endonuclease, they are not cut and remain in high molecular form. After digestion of the total DNA, such undigested “spared” sequences are found at the top of the gel and have been referred to as “relic” DNA. Repeated digestion of DNA with restriction enzymes followed by steep, linear sucrose gradient centrifugation provides a method for enrichment of the spared, repeated sequences. Various restriction endonuclease spared fractions of rye were purified by velocity sedimentation. Each of the Hind III, Eco RI, Bam HI, and Bgl II digests of Secale cereale and S. silvestre, DNA showed a higher proportion of “relic” DNA in S. cereale. “Hind III relic” in the two species were 5 and 2.3% of the total DNA of S. cereale and S. silvestre respectively.
DPYD and TYMS polymorphisms as predictors of 5 fluorouracil toxicity in colorectal cancer patients
Published in Journal of Chemotherapy, 2023
Yassine Khalij, Imtinen Belaid, Sana Chouchane, Dorra Amor, Asma Omezzine, Nabila Ben rejeb, Slim ben Ahmed, Ali Bouslama
The study of genetic polymorphisms focused on the DPYD and TYMS genes. We carried out DNA extraction from whole blood leukocytes taken from EDTA using the salting-out method and genotyping by direct PCR for TYMS 5′UTR VNTR and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method, using specific restriction enzymes (Table A.1). We used the following PCR protocols for all 8 SNPs: first denaturation, 5 min; at 95 °C, denaturation 45 s; at 95 °C, hybridization 45 s (see Table A.1 for temperature), elongation 1 min; at 72 °C, final elongation 7 min; and at 72 °C for 32 cycles. We used DreamTaq Green DNA Polymerase (5 U/µL, Thermofisher; EP0712) following the recommended PCR protocol. The digestion reactions with restriction endonuclease contained 1X buffer, 5 U of RE (restriction enzyme), and 3 µl of PCR-amplified DNA. We incubated the restriction endonuclease digestions at the temperature recommended by the manufacturer (Table A.1).
Association of CFI, IL-8, TF and TFR2 Genetic Polymorphisms with Age-Related Macular Degeneration in a Northeastern Chinese Population
Published in Current Eye Research, 2022
Zhi-Yu Xu, Jing-Fan Gao, Lu Zhang
Peripheral venous blood was extracted and collected with a blood genomic DNA Kit (Beijing Tian-gen Biotechnology Company), and the genomic DNA was labeled and stored in a constant-temperature freezer at −20 °C. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was used for genotyping the 5 SNPs. The amplification reaction was carried out in a PCR machine. An agarose gel was prepared, and 8 µl of ethidium bromide (EB) dye was added and mixed thoroughly. The PCR product was electrophoresed on the gel at a constant 100 V for target band detection. The endonuclease digestion system was as follows: PCR product 5 µl, CutSmart buffer 1 µl, ddH2O 3.7 µl, and restriction endonuclease 0.3 µl. The 10 µl enzyme digestion reaction system was mixed well and then placed in a constant-temperature incubator. We prepared a 3% agarose gel with 8 µl of ethidium bromide, 7 µl digested product was added to each lane, and after electrophoresis, the gel was placed in a gel imaging analysis system (Alpha Innotech, USA) to analyze the product bands after restriction enzyme digestion and genotyping. The primer sequences and restriction endonucleases are shown in Table 1.
A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction
Published in Xenobiotica, 2021
Han Li, Yu-Guang Wang, Zeng-Chun Ma, Gao Yun-Hang, Song Ling, Chen Teng-Fei, Zhang Guang-Ping, Yue Gao
3-Methylcholanthrene (3-MC), α-naphthoflavone (α-NF), β-naphthoflavone (β-NF), indirubin (IND), 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl oxime (CITCO), phenobarbital sodium salt (PB), clotrimazole (CLOT), dexamethasone (DEX), nifedipine (NIF), omeprazole (OMEP), rifampicin (RIF), dimethylsulphoxide (DMSO), and G418 were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was purchased from Cerilliant Co. (Round Rock, TX, USA). Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). Restriction endonucleases and T4 DNA ligase were purchased from TaKaRa Biotechnology (Dalian, China). The pcDNA3.1 vector was purchased from Promega (Madison, WI, USA) and the pGLuc-basic2 vector was purchased from New England Biolabs (Beijing) Ltd. (Beijing, China). The TransScript™ First-Step RT-PCR Super Mix and SYBR® Green Master Mix were purchased from TransGen Biotech Co. Ltd (Beijing, China). An antibody against GAPDH and an anti-rabbit IgG HRP-linked antibody were purchased from Cell Signalling Technology (Danvers, MA, USA). The antibodies against CYP1A1, CYP2B6, CYP3A4, AhR, CAR, and PXR were purchased from ProteinTech (Rosemont, IL, USA).