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Genetics and exercise: an introduction
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Claude Bouchard, Henning Wackerhage
One of the greatest scientific triumphs of the late 20th and early 21st centuries was the sequencing of the human genome and of the genomes of many other species. The starting point of all of these DNA sequencing projects was the development of DNA sequencing using the chain-termination method by the twice Nobel Laureate, Frederick Sanger (18). This method was first used to sequence all the 16,569 base pairs of the human mitochondrial genome (mitochondria have their own DNA termed mtDNA) (19). Following this, the state-funded multi-national Human Genome Project consortium ended up competing with a privately funded effort (Celera Corporation), the latter using so-called shotgun DNA sequencing. Both teams published their draft genome sequences in 2001, first the Human Genome Project consortium in Nature on the 15th of February (20) followed by Craig Venter and Celera Corporation one day later in Science (21).
History and First Look
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
Summarizing, the application of the phage RNA messengers (i) verified the fidelity of the in vitro translation system, (ii) contributed crucially to the mechanisms of chain initiation and chain termination, including determination of the mechanism of nonsense suppression and identification of punctuation signals in protein synthesis, which were not readily studied by the use of synthetic polyribonucleotides, (iii) initiated studies of ribosome recognition by correct polypeptide initiation sites on mRNA, and (iv) established therefore a solid basis for the modern understanding of protein biosynthesis, as reviewed substantially by Lucas-Lenard and Lipmann (1971). Moreover, the sequences of the phage RNAs have confirmed the strong accuracy of the genetic code.
Gastrointestinal cancer
Published in Michael JG Farthing, Anne B Ballinger, Drug Therapy for Gastrointestinal and Liver Diseases, 2019
Justin S Waters, David Cunningham
A more recently developed antimetabolite drug that is proving very useful, particularly in carcinoma of the pancreas, is gemcitabine. This is a cytidine analogue that is incorporated into DNA in its triphosphate form, after activation by deoxycytidine kinase. This results in chain termination after the addition of one further nucleotide. It also inhibits the activity of several enzymes involved in cytidine metabolism, including ribonucleotide reductase, dCMP deaminase, and CTP synthetase, and is incorporated into RNA, leading to inhibition of RNA synthesis. Cell cycle arrest occurs in the S phase followed by the induction of cell death by apoptosis.
Autoimmune disorders associated with common variable immunodeficiency: prediction, diagnosis, and treatment
Published in Expert Review of Clinical Immunology, 2022
Niloufar Yazdanpanah, Nima Rezaei
Genetic evaluation is helpful to confirm CVID diagnosis in many cases. DNA sequencing via dideoxy chain termination, also known as first-generation sequencing, is considered as the gold standard for mutation screening. Copy number variant (CNV) analysis has been used to explore the genetic basis in CVID [169,170]. In patients that a single specific gene or a set of genes are suspected, Sanger sequencing is helpful. In addition, Sanger sequencing is the diagnosis method of choice for confirmation of the results obtained from the high throughput sequencing methods [13]. Moreover, Sanger sequencing is commonly performed to search for a specific mutated gene in family members of a proband with a confirmed mutation (segregation analysis) [13]. Next-generation sequencing (NGS) includes targeted gene sequencing (TGS), whole exome sequencing (WES), and whole genome sequencing (WGS). Considering that the majority of known monogenic defects leading to CVID-like manifestations were recognized sporadically in individual patients rather than families, it is not recommended to use TGS in a patient with a complicated CVID-like phenotype that is suspected to carry a novel genetic variant [125]. TGS explores a set of multiple genes, while WES explores only the protein-coding part of the genome, in which 85% of the disease-causing variants are located [171]. WGS, which is not commonly used in CVID clinical practice, explores both protein-coding and non-coding parts of the genome and is recommended when TGS and WES fail to detect the mutated variant [172].
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].
ID CORE XT as a tool for molecular red blood cell typing
Published in Expert Review of Molecular Diagnostics, 2019
Carolina Bonet Bub, Lilian Castilho
Modern sequencing technology is gaining space and is used in some immunohematology laboratories, but it is still very expensive. There are several procedures. The oldest method, available since the 1980s, is the Sanger sequencing, a chain-termination sequencing technique that uses an enzymatic degradation procedure and whose results are read by capillary electrophoresis. Also available is the new generation sequencing technology (NGS), which allows obtaining information from multiple genes and in a single race. The first NGS technology to be released in 2005 was the pyrosequencing method by 454 Life Sciences (now Roche) [24]. There are currently different platforms, with different analysis capabilities and reaction times. This technology has some known limitations, such as the large amount of data generated and the fact that it often has a little-known clinical significance, as well as the high levels of homology between some blood group genes [25–27]. However, recent studies showed a 99.8% concordance across 90 genomes and 99.9% after improvement of the software algorithm for blood group typing, reinforcing the reliability of this technology [28].