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Genomic Informatics in the Healthcare System
Published in Salvatore Volpe, Health Informatics, 2022
DNA is the code of all biological life on earth. Humans have sought to unravel its mysteries so that the origins of life itself may be revealed. The first sequencing methodology, known as Sanger sequencing, uses specifically manipulated nucleotides to read through a DNA template during DNA synthesis. This sequencing technology requires a specific primer to start the read at a specific location along the DNA template and record the different labels for each nucleotide within the sequence up to 1000–1200 base pairs (bps). Subsequently, an approach called shotgun sequencing was developed for longer read of sequences. In this approach, genomic DNA is enzymatically or mechanically broken down into smaller fragments and cloned into sequencing vectors in which cloned DNA fragments can be sequenced individually. The complete sequence of a long DNA fragment can be eventually generated by these methods by alignment and reassembly of sequence fragments based on partial sequence overlaps.
Clinical genetics
Published in C. Simon Herrington, Muir's Textbook of Pathology, 2020
Until recently, the majority of diagnostic DNA analysis was carried out using PCR (polymerase chain reaction) amplification and Sanger sequencing, also known as the ‘dideoxy’ or ‘chain termination’ method. This technique was developed by Frederick Sanger in 1977, and allows sequencing of short stretches of DNA, up to approximately 700 base pairs (bp) in length. Analysis of a gene using this technology requires amplification of each part of the gene in suitable fragments, with sequencing of each. The high overall cost of this restricts the amount of analysis that can be performed. To use this effectively in the diagnostic setting, it is necessary to identify which gene requires analysis from the clinical phenotype. Sanger sequencing is still the method of choice for analysis of very specific genetic regions, e.g. to test a family member to see if he or she carries a mutation found in other relatives.
Genetics
Published in Stephan Strobel, Lewis Spitz, Stephen D. Marks, Great Ormond Street Handbook of Paediatrics, 2019
Jane A. Hurst, Richard H. Scott
Since the 1980s, the mainstay of DNA sequencing has been Sanger sequencing, a technique which requires PCR amplification of the target gene in small fragments followed by sequencing of the amplified fragment. This is a highly accurate but labour intensive and therefore expensive technique. Sanger sequencing does not usually detect larger-scale copy number alterations. For many genes, comprehensive mutation testing therefore also requires the use of a copy number analysis technique such as multiplex ligation-dependent probe amplification (MLPA).
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].
STOX1 promotor region -922 T > C polymorphism is associated with Early-Onset preeclampsia
Published in Journal of Obstetrics and Gynaecology, 2022
Seyda Akin, Ergun Pinarbasi, Aslihan Esra Bildirici, Nilgun Cekin
DNA sequencing was conducted by Sanger sequencing method. A 3–5-μL aliquot of PCR product (collected directly or from re-amplification of excised SSCA bands) was used in a standard protocol for fluorescently labelled dideoxy-nucleotides (BigDye, Applied Biosystems, Life Technologies), with injection into a capillary electrophoresis instrument (ABI 3500, Life Technologies) for separation and detection. The sequences obtained were compared with the reference sequence NC 000017 (www.ncbi.nlm.nih.gov), and deviations were recorded as mutations or polymorphisms. Chromas Lite 2.6.6 software was used to display the results and to scan the changes in the array. Also, DNA sequencing was employed using the next generation sequencing system Illumina technology. IGV 2.6.3 (Archived) program was utilised to display the results and to observe the changes in the array.
T cell receptor revision and immune repertoire changes in autoimmune diseases
Published in International Reviews of Immunology, 2022
Ronghua Song, Xi Jia, Jing Zhao, Peng Du, Jin-an Zhang
The limited understanding of autoantigens and autoreactive TCRs once stopped people from further exploration of AIDs. Analyzing TCR repertoire is a very useful approach, but is also challenging due to the large number and highly variable nature of TCR repertoire. Despite deciphering the entire T-cell repertoire in diseases is still far away, important progresses have been made to bring us closer to this goal. Scientists have tried a variety of methods to detect the TCR library, such as Sanger sequencing, flow cytometry, PCR for CDR3 region, and Southern blot for TCR DNA [47–50], but all these methods have their own limitations. Sanger sequencing can only sequence up to 700 bp nucleic acid, and its low-throughput makes it a laborious method with high failure rate [49]. Flow cytometry has the advantage of assessing actual protein expression on cell surface, but it can only reveal the frequency of V-family usage [48]. Multiplex PCR, an RNA (cDNA)-based approach was also used in TCR detection [50]. However, this method can hardly make precise and credible comparisons between samples, even when using a complex scoring system that defines the heights of clonal peaks relative to the remaining repertoire [50,51]. Additionally, the unavoidable PCR bias, caused by differences in primer annealing and amplification conditions, also hindered its application [51]. Southern blot is currently not used anymore, because it requires large amounts of DNA and is not effective or accurate [47].