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Cancer Informatics
Published in Trevor F. Cox, Medical Statistics for Cancer Studies, 2022
In Sanger sequencing, four inhibitors of chain elongation in the process of DNA polymerisation are used. They are dideoxynucleotides (modified nucleotides), denoted by ddATP, ddCTP, ddGTP, ddTTP, and they stop a chain from further elongation at an A nucleotide (denoted dATP), a G nucleotide (dGTP), a C nucleotide (dCTP) and a T nucleotide (dTTP) respectively. Polymerase chain reaction is carried out with four separate samples of the DNA as described above, but with one of dideoxynucleotides added to each sample. So the first sample might have ddCTP added but in a smaller concentration than the “C” nucleotide, and similarly for the other three samples. During PCR for the strand (or fragrant) building caused by the polymerase in the first sample, each fragment will build as normal with the dATPs, dCTPs, dGTPs, dTTPs being added until, at random, a ddCTP is added and so the fragment will terminate at that point, and similarly for the other three samples. After the PCR is finished, each of the four samples will contain fragments of various lengths, all terminating in their added dideoxynucleotide.
Preimplantation Genetic Diagnosis for Single Gene Disorders
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Ana Cervero, Jose Antonio Martínez-Conejero, Lucía Sanz-Salvador, Claudia Gil-Sanchís, Maribel Sánchez-Piris, Laura Iñiguez Quiles
Genotyping of the amplified products can be performed by means of different strategies, with minisequencing the most frequently used method for the detection of point mutations (23). In the minisequencing technique, a primer extension reaction is performed, allowing rapid and accurate detection of point mutations. The minisequencing primer is designed to anneal one base before the target site, and it can only be elongated with one specific dideoxynucleotide. The four different dideoxynucleotides are labeled with different fluorochromes, and the products can be analyzed on an automated DNA sequencing system. Other strategies such as amplification refractory mutation system (24), restriction enzyme digestion (25), and real-time PCR (26) have been also applied in PGT-M. Small deletions and duplications can also be detected by sizing of PCR products from specific regions containing the mutation under study.
Molecular Genetics and Diagnostic Testing
Published in Merlin G. Butler, F. John Meaney, Genetics of Developmental Disabilities, 2019
Determining the order of the nucleotide bases in DNA is typically done by the dideoxy method (chain-termination of Sanger method) (10). DNA polymerase proceeds to synthesize DNA strands of various lengths, stopping DNA replication at one of four bases and determining the resulting fragment lengths. In addition to the DNA template and the enzyme, sequencing reactions contain a primer to bind to the DNA, four deoxynucleotides to be incorporated into the new DNA strand, one labeled deoxynucleotide (using radioactivity or fluorescent dye), and one dideoxynucleotide that terminates the strand wherever it is incorporated. The concentration of the nucleotides is adjusted, so that dideoxynucleotides are incorporated into each position in which that nucleotide occurs producing a collection of DNA fragments, each with a dideoxynucelotide at the end. The fragments are separated by electrophoresis and the positions of the nucleotides visualized by autoradiography or by an automated laser-equipped instrument to determine the sequence (Fig. 7).
Cataract in You-Hoover-Fong syndrome: TELO2 deficiency
Published in Ophthalmic Genetics, 2020
Cristina Del-Prado-Sánchez, Judith Armstrong-Moron, Carla Veiga, Stefano Grixolli-Mazzon, Àngels García-Cazorla, Natalia Juliá-Palacios, Marta Morales-Ballús
A massive sequencing of the DNA was performed to find the cause of their developmental delay, microcephaly, and cataracts. DNA quality was determined by continued lecture of the optical density using Nanodrop equipment. Integrity was checked up by electrophoresis and stain with SYBRGreen II. Capture and massive sequencing of the exons of the human genes was performed using MedExome assay with a mean coverage of, at least, 30x. Confirmation of the candidate variants was conducted using an independent sequencing method, direct sequencing Sanger technique. DNA fragment carrier of the variant subject of study is amplified by PCR. This fragment is subdued to an additional PCR reaction with dideoxynucleotides marked with fluorescein that are analyzed by ABI3100 equipment. Bioinformatic analysis of massive sequencing data was performed to identify unique nucleotide variants and small insertions or deletions in comparison with the reference genome (NCB1 v37 version). Filtering of the variants was made using the following parameters: minimum coverage 6x, maximum coverage 200x, bias on the DNA chain sequenced >-0.10, the number of sequences with the variant (for heterozygosis) >20%, maximum length for variant allele homopolymers 8.
Common mutations of interest in the diagnosis of amyotrophic lateral sclerosis: how common are common mutations in ALS genes?
Published in Expert Review of Molecular Diagnostics, 2020
Benedetta Perrone, Francesca Luisa Conforti
Genetic testing for SOD1, TARDBP, and FUS genes includes first and second-generation DNA sequencing methods. Sanger sequencing is the ‘first-generation’ DNA sequencing method, widely used in ALS diagnosis, first emerged in 1977 [110]. Sanger Sequencing is known as the chain termination or the dideoxynucleotide or the sequencing by synthesis method. It consists of using one strand of the double-stranded DNA as a template to be sequenced. This sequencing is made using chemically modified nucleotides called dideoxynucleotides (dNTPs). These dNTPs marked for each DNA bases by ddG, ddA, ddT, and ddC also include a fluorescent marker (A is indicated by green fluorescence, T by red, G by black, and C by blue). The fluorescent dideoxynucleotides (dNTPs) are used for elongation of nucleotide, once incorporated into the DNA strand they prevent the further elongation. Then, we obtain DNA fragments ended by a dNTP with different sizes and fragments, separated according to their size by capillary electrophoresis. A laser within the automated machine used to read the sequence detects a fluorescent intensity that is translated into a ‘peak’ revealing heterozygous or homozygous variants within a sequence [111].
Radiotherapy in combination with hyperthermia suppresses lung cancer progression via increased NR4A3 and KLF11 expression
Published in International Journal of Radiation Biology, 2019
Beomseok Son, Jaewan Jeon, Sungmin Lee, Hyunwoo Kim, Hyunkoo Kang, HyeSook Youn, Sunmi Jo, BuHyun Youn
In the present study, we performed global gene expression analyses using Affymetrix GeneChip® Human Gene 2.0 ST oligonucleotide arrays. Total RNA was isolated using Trizol reagent (Bioline, London, UK). RNA quality was assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands), and its quantity was determined using an ND-2000 spectrophotometer (Thermo Inc., DE, USA). For each RNA sample, 300 ng was used as an input into the Affymetrix procedure as recommended by the protocol (http://www.affymetrix.com). Briefly, 300 ng of total RNA from each sample was converted to double-stranded cDNA. Using a random hexamer incorporating a T7 promoter, amplified RNA (cRNA) was generated from the double-stranded cDNA template through an in vitro transcription (IVT) reaction and purified with the Affymetrix sample cleanup module. cDNA was regenerated through random-primed reverse transcription using a dNTP mix containing dUTP. The cDNA was then fragmented by UDG and APE 1 restriction endonucleases and end-labeled by a terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented end-labeled cDNA was hybridized to the GeneChip Human Gene 2.0 ST arrays for 16 h at 45 °C and 60 rpm as described in Gene Chip Whole Transcript Sense Target Labeling Assay Manual (Affymetrix). After hybridization, the chips were stained using streptavidin phycoerythrin (SAPE), washed in a Genechip Fluidics Station 450 (Affymetrix), and scanned using a Genechip Array scanner 3000 7 G (Affymetrix).