DNA Sequence

However, modern DNA sequence-based technology now facilitates HLA typing at allele (2-field) level, which together with knowledge of tertiary protein structure enables a new approach to assess mismatched alloantigen immunogenicity.

From: Kidney Transplantation - Principles and Practice (Eighth Edition), 2019

Chapters and Articles

DNA SEQUENCING

K.R. Mitchelson, in Encyclopedia of Analytical Science (Second Edition), 2005

Introduction

DNA sequencing is very big business. Approximately US$3 billion was spent in 2003 on sequencing reagents and enzymes, and on the analyzer equipment and software for automated sequence acquisition. The majority of this sequence output was determined using capillary electrophoresis (CE) technology, which has commensurately developed rapidly over the past 10 years. CE offers high resolution and high throughput, automatic operation, and data acquisition, with online detection of dyes bound to DNA extension products. Operational advances such as pulsed-field and graduated electric fields and automated thermal ramping programs as the run progresses result in higher base resolution and longer sequence reads. Advanced base-calling algorithms and DNA marker additives that utilize known fragment sizing landmarks can also help to improve fragment base-calling, increasing call accuracy and read lengths by 20–30%. Despite the high efficiency of CE sequencers, the complete delineation of the human genome and its implication for genome-wide analysis for personalized medicine is driving the development of devices and chemistries capable of massively increased sequence throughput, compared to the conventional CE sequencers. Miniaturization of CE onto chip-based devices provides all of the above facilities – a significant improvement in the speed and improved automation of analysis. New array-based sequencing devices also promise a quantum increase in efficiency. Each of these new devices provides an extremely high throughput, high-quality-data, and low-process costs. This article also examines the automation and improvement of sequencing processes, DNA amplification processes, and alternative approaches to sequencing.

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Analysis of Human Genetic Variations Using DNA Sequencing

Gregory A. Hawkins, in Basic Science Methods for Clinical Researchers, 2017

Bioinformatic Analysis of NGS Data

The strength of NGS compared to Sanger sequencing is the ability of NGS to analyze a large panel of genes, or a complete genome, in a single assay. Unlike Sanger sequencing, however, NGS requires powerful computing and data storage resources to handle the large data files produced during a single analysis. There are numerous commercial software programs that can be purchased for analysis of NGS data; however, many of the best bioinformatics programs can be acquired for free. Much of the free NGS analysis software do not have graphical user interfaces (GUI) and thus experience with “command line” programing may be necessary. It is advisable to have a strong bioinformatics resource before attempting NGS data analysis.

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Gastrointestinal Peptides

Celia Chao, Mark R. Hellmich, in Physiology of the Gastrointestinal Tract (Fifth Edition), 2012

6.2.6.1 Cloning

The cDNA sequences for both the rat CCK1 receptor128 and dog gastrin receptor129 were first reported in early 1992. Later that same year, CCK2 receptor cDNA clones were isolated from the rat brain and the rat pancreatic acinar carcinoma cell line, AR4-2J, using a low-stringency hybridization procedure with a CCK1 receptor probe.130 Comparison of the dog gastrin receptor and rat CCK2 receptor cDNA sequences, as well as their binding and signaling properties, demonstrated that they were interspecies homologs of the same receptor subtype. Subsequently, the human CCK2 receptor cDNA was cloned131 and its genomic DNA sequence determined.132

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MICROBIOLOGY | Detection of Foodborne Pathogens and their Toxins

J.W. Austin, F.J. Pagotto, in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

DNA Sequencing

Automated DNA sequencing procedures now allow rapid sequencing of extended DNA sequences. This has facilitated sequencing of entire genomes, including the human genome. Perhaps the ultimate bacterial identification procedure of the future will be genome sequencing. Certainly, current technologies allow for rapid amplification and sequencing of the genes, such as the 16S rRNA of bacterial isolates. Because 16S rRNA genes are conserved among isolates of the same species, yet vary between species, phylogenetic trees describing their evolutionary relationships have been described. Indeed, the universal phylogenetic tree describing the phylogeny of the living world has been based on 16S or 18S rRNA comparative sequence analysis.

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Parentage Testing and Kinship Analysis

R.W. Allen, J. Pritchard, in Encyclopedia of Forensic and Legal Medicine (Second Edition), 2016

DNA Sequencing

Next generation DNA sequencing (NGS) may soon have a role in relationship and forensic testing. Massively parallel DNA sequencing can reveal the nucleotide sequences of thousands of individual template DNA molecules within a given sample simultaneously. The variation that characterizes an individual sample is far beyond what is revealed through traditional Sanger sequencing, whether the sample is from a single source sample or a sample made up of DNAs from various individuals, such as might be found in an evidentiary sample. NGS is not only used to characterize nuclear DNA and RNA but also has applications for mtDNA analysis that focuses on the entire mitochondrial genome rather than just specific hypervariable regions (HVI and II). In addition, since the template size suited for NGS ranges from 200–400 basepairs, NGS is well suited to provide DNA typing information with badly degraded genomic DNA such as might be recovered from human remains.

In rare cases where monozygotic twins are tested as the alleged father or suspects in a forensic case, it has been demonstrated that NGS can be used to determine single nucleotide differences that can distinguish between ‘identical’ twins (Karow, 2013). Mutations that distinguish the twins occur just before or after the morula splits. In order to be transmitted to a child, they need to be present in the father’s germline. Samples used for the testing has included semen, blood, and buccal swabs; slight differences in the number of informative SNPs occurs in the various tissues, possibly due to mutations that continue to arise during the development of an embryo, with some present in the germline but not in other tissues and vice versa. Samples from both twins must be tested so that the SNPs differing in the twins can be compared to evidence collected at the crime scene. Because of the high error rate of NGS, confirmation of the difference(s) must be confirmed with traditional Sanger sequencing. It is expected that there would be no issues with introducing NGS in court because Sanger sequencing, a well-established method, is used to confirm the findings.

Because of the high number of missing persons cases and mass disasters, NGS is also finding use when mtDNA testing is used. Whole genome sequencing that increases the discrimination power of traditional mtDNA testing is required to identify individuals. It is also a higher throughput method than traditional Sanger sequencing and provides the opportunity to deconvolute mixtures when material contains DNA from more than one individual. It is expected that NGS will replace Sanger sequencing for all but a few forensic mtDNA cases in the next 5 years (Melton, 2014).

A number of NGS kits have recently been developed that can simultaneously amplify autosomal, mitochondrial, and Y-STRs in combination with single nucleotide repeats (SNPs). This substantially increases the amount of data that can be obtained from a single sample but still allows compatibility with established criminal databases such as Combined DNA Index System (CODIS). Although traditional STR typing using capillary electrophoresis is the more cost-effective method, NGS can extend the capabilities of a laboratory to obtain DNA profiles from compromised samples and can provide additional information, such as probable ancestry and physical features of the individual whose DNA is being tested. These kits or similar ones could also be used in relationship testing laboratories, particularly when the sample is a nontraditional one (i.e., not blood or a buccal swab). There are laboratories that are currently using a noninvastive prenatal paternity test (Ryan et al., 2013). Fetal cell-free DNA (cfDNA) typically comprises <20% of the total cfDNA in maternal plasma; cfDNA is highly fragmented. SNP microarrays or NGS has been used to characterize the fetal cfDNA and compare it to the DNA from an alleged father and a mother in a prenatal parentage test. Aggregating data from over 300 000 SNPs allows for highly accurate paternity determinations. The ability to separate out the fetal cfDNA can also allow for testing for chromosomal abnormalities using NGS (Avent, 2012).

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Patenting of Life Forms

Padma Nambisan, in An Introduction to Ethical, Safety and Intellectual Property Rights Issues in Biotechnology, 2017

14.4.4 Sequences Used in Production of Therapeutic Proteins

First generation gene patents were issued for full length DNA sequences that made proteins of use in medicine and were valuable as they helped recoup the cost of the research and development. The Nuffield Council on Bioethics (2002) does however recommend that these patents should be narrowly defined so that the rights to the DNA sequence extend only to the protein described.

Key Takeaways

Patenting genes and DNA sequences:

Sequences used for diagnostic testing: isolated DNA sequences are not patentable if similar to that in the living organism; cDNA is patentable

Sequences used as research tools: ESTs and SNPs are not patentable if “substantial, credible and specific use” is not demonstrated

Sequences used in gene therapy: Nuffield Council on Bioethics recommends that protection by product patents should seldom be permitted for DNA sequences used in gene therapy

Sequences used in production of therapeutic proteins: most patented class of DNA sequences

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Chromosome Rearrangements and Translocations

Stefan K. Bohlander, ... Alix Coysh, in Encyclopedia of Cancer (Third Edition), 2019

DNA sequencing methods

DNA sequencing techniques determine the precise order of nucleotides in a DNA molecule. Whole-genome sequencing (WGS) uses a number of next-generation sequencing (NGS) methods that sequence entire genomes and they may thus be used to identify structural rearrangements across a genome. For the detection of structural rearrangements, the most efficacious WGS NGS approach is to combine mate pair sequencing and short-insert paired-end sequencing.

In mate pair sequencing, DNA is sheared into fragments of a desired length, often between 2 and 5 kbp. However this poses an issue because it is not feasible to sequence long fragments over 1 kbp using NGS. To combat this, the long fragments are biotinylated at their ends and circularized, and then the DNA rings are fragmented into approximately 400–600 bp. Next the biotin-containing fragments are extracted and the result is fragments short enough to be sequenced which still contain the two ends of the initial long fragment. Because the distance between these two ends was between 2 and 5 kbp depending on the original fragmentation parameters, if they are sequenced and mapped to a reference genome and found to be much closer together or further apart than they should be or even map to different chromosomes, then this indicates that a structural rearrangement has taken place.

In short-insert paired-end sequencing, DNA is fragmented into much shorter insert sizes between 200 and 800 bp, and adapter molecules containing primers are attached to the ends of the fragmented molecules. Because the primers in the adapters are different at each end, this allows bi-directional sequencing of the DNA fragments, which provides greater sequencing accuracy than sequencing from a single end because it generates greater coverage when aligned to a reference genome.

Both of these methodologies complement one another in the detection and evaluation of structural rearrangements. This is because mate pair Sequencing enables more comprehensive detection of large structural rearrangements, and paired-end sequencing provides more detailed sequencing data filling in any gaps that may be present in the mate pair sequencing genome coverage, as well as being particularly useful in highly repetitive regions. When the data from each of these DNA sequencing methods is analyzed together it facilitates determination of: the genomic loci of structural rearrangements, the size of the rearrangements, the altered DNA sequence following each rearrangement, the genes involved in each rearrangement, and an estimation of the impact of each rearrangement. Together, this gives WGS methods an advantage of having unprecedented sensitivity and resolution of structural and mutational changes. However, it should be noted that mate-pair sequencing is technically challenging and that WGS for the detection of structural rearrangements in tumor samples is a costly and highly experimental approach at present and far from being used routinely.

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Lipoprotein and Lipid Metabolism

Robert A. Hegele, in Emery and Rimoin's Principles and Practice of Medical Genetics (Sixth Edition), 2013

96.7.2.1 Clinical Features

DNA sequencing of individuals with very high plasma HDL cholesterol identified a family with a missense mutation in SCARB1, altering the amino acid sequence: P297S (113). Heterozygotes had mean HDL cholesterol that was increased by ~50% over noncarriers. The lipid profile was otherwise normal, as was CVD risk. Platelets from carriers had increased unesterified cholesterol content and impaired function. In addition, adrenal steroidogenesis was attenuated, with decreased urinary excretion of sterol metabolites, decreased response to corticotropin stimulation, and symptoms of diminished adrenal function (113).

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Mycobacterium tuberculosis

Silvia S. Chiang, Jeffrey R. Starke, in Principles and Practice of Pediatric Infectious Diseases (Fifth Edition), 2018

Genetic Sequencing.

DNA sequencing, which has been used to identify M. tuberculosis strains for epidemiologic purposes, has been adapted for DST. Unlike NAATs, which use probes to detect a set number of resistance-conferring mutations fixed a priori into the product design, sequencing platforms can identify all possible mutations in target genes and be rapidly modified to add more genes. Sequencing can also distinguish between silent and phenotypically significant mutations. Studies have shown more than 90% sensitivity and specificity for isoniazid resistance and more than 95% sensitivity and specificity for rifampin resistance.184,187–189

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Cell Signaling Events

In Medical Cell Biology (Third Edition), 2008

Neuregulin

The RAS/MAPK signal transduction pathway is a major signal transduction pathway in vertebrate cell-to-cell communication and has been co-opted by numerous signaling molecules and their receptors to effect their signal transmission (Fig. 8-2). As with the catalytic tyrosine kinase receptors and their signal transduction pathways, the RAS/MAPK pathway can be directed to serve different biological ends by hooking up with different receptor-ligand pairs. An example of this can be seen in heart development. Cardiac cells are derived from progenitor cells within the mesoderm of the developing blastula. The induction of cardiac cells from mesodermal precursors is dependent on their receiving signals from the neighboring endodermal germ cell layer. Numerous studies have shown that a critical signal for this process is FGF. In those cells of the mesoderm expressing FGFRs, the endoderm-derived FGF signal activates their RAS/MAPK pathway as part of the process of inducing the cardiac cell lineage. Later in development, more differentiated heart cells express a new array of cell-surface receptors that direct the RAS/MAPK pathway toward different biological ends. A prime example of this is seen in the cell-to-cell signaling underlying the process of trabeculation. Trabeculation is the extension and involuting of the compact myocardial layer of the nascent ventricular chamber into the chamber space, a process that helps the heart maintain blood flow before the heart myocardium is able to contract on its own. In this case, a signaling molecule called neuregulin-1 (NRG1) binds to Erb receptor tyrosine kinases on the surface of myocardial cells and activates the RAS/MAPK pathway (see Fig. 8-2). Unlike the earlier developmental signaling events between cells of relatively undifferentiated germ cell layers, NRG signaling provides an interesting example of how one differentiated cell type can direct the activity of another differentiated cell type within a nearly functional organ, such as the late embryonic heart. In this case, the NRG1 signal originates in the endocardial cell layer of the heart and signals to cells in the myocardial layer of the heart to form the trabeculated myocardium (Fig. 8-3).

Figure 8-2. ErbB-induced signaling pathways. Ligand binding induces receptor homodimerization or heterodimerization, which leads to activation of tyrosine kinase activity and the phosphorylation of specific tyrosine residues (pY) within the carboxyl-terminal tail of ErbBs. Signaling effectors that contain either the SH2 or phosphotyrosine binding (PTB) domains for binding pY-containing peptides are recruited to activated receptors and direct the ErbB signal down specific signaling pathways. These pathways include the RAS-mitogen–activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)-Akt, phospholipase C-protein kinase C (PLC-PKC), and the Jak/Stat signaling pathways. Virtually all ErbB receptors signal through the RAS/MAPK signal transduction pathway, which is shown.

(Modified from Marmor MD, et al. Int J Radiat Oncol Biol Phys 2004;58:903, by permission.)Copyright © 2004

Figure 8-3. Loss of neuregulin-1 signaling through the ErbB2/ErbB4 heterodimer leads to defective trabeculation of the ventricles during heart development in embryonic day 10.5 mice. A: Heart section from a neuregulin knockout mouse, embryonic day 10.5. B: Heart section from a wild-type mouse, embryonic day 10.5. M, myocardium; OT, outflow tract; P, pericardium; T, trabeculated myocardium; V, ventricle. Asterisks denote cardiac cushions. C: Neuregulin-1 signaling from endocardium (blue) to ErbB2/ErbB4 heterodimers in the myocardium (red).

(From Garratt AN, et al. Trends Cardiovasc Med 2003;13:80, by permission.)Copyright © 2003

In the heart, NRG1 signals to responsive cells via ErbB receptors that are heterodimers of ErbB2 and ErbB4, two of the possible four ErbB receptors encoded in vertebrate genomes (Fig. 8-4). ErbB2 is a “ligandless” receptor that is a heterodimerizing partner with ErbB receptors that can bind ligand. In this case, specificity for NRG1 is likely to be in the ErbB4 receptor. ErbB receptors contain an extracellular ligand-binding domain and a single hydrophobic transmembrane domain. The intracellular portion of ErbB receptors consists of a highly conserved tyrosine kinase domain. Ligand binding by ErbB receptors induces either formation of a homodimer of the same receptor type or a heterodimer of different receptor types. In either case, the dimerization leads to phosphorylation of tyrosine residues on the partner receptor, resulting in an increase in its kinase activity. Additional tyrosine phosphorylation on residues within the carboxyl-terminal tail of the receptors enables the recruitment and activation of adaptor proteins containing Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains (see Fig. 8-2). Proteins that contain these structural features are recruited to activated receptors where they assemble into multiprotein complexes that initiate ligand signaling to downstream signal transduction pathways. Binding of the activated receptor by these adaptor proteins comprises the first step in the intracellular signaling cascade and, as such, directly determines which of the various downstream signal transduction pathways will be used by the activated receptor to transduce the signal. In the case of ErbB signaling, this may be any of four different signal transduction pathways: the RAS/MAPK pathway, the PI3K-protein kinase B (PKB)/Akt pathway, the phospholipase C-protein kinase C (PLC-PKC) pathway, or the Jak/STAT pathway (see Fig. 8-2). For formation of trabeculae, the signal transduction pathway used by the ErbB2/4 receptor has not yet been formally demonstrated. What is known is that all ErbB ligands and receptors can activate the RAS/MAPK pathway, so in all likelihood this pathway plays a role in NRG1 signaling to myocardial cells. Signaling by this pathway begins with the binding of NRG1 to the ErbB2/4 receptor and the crossphosphorylation of tyrosine residues in the intracellular domain of the receptors by the kinase activity of the receptors themselves. This has a twofold purpose, the first being to increase kinase activity of the receptors, and the second to create a conformational domain for the recruitment and binding of the RAS/MAPK effector complex of proteins. This complex links ErbB receptors to the RAS signal transduction pathway by bringing activators of RAS into close proximity with RAS to effect its activation. The major components of this complex are Grb2, an adaptor protein, and Sos, a guanine nucleotide exchange factor that is the actual activator of RAS. Grb2 and Sos form a complex that is recruited to phosphorylated ErbB receptors through binding of the SH2 domain of Grb2 to specific phosphotyrosine sites of the receptor. This brings Sos into close proximity with RAS at the plasma membrane where it activates RAS by exchanging a GDP nucleotide (RAS bound to GDP is inactive) for GTP, the nucleotide that binds and activates RAS. Once activated, RAS binds to and activates the Raf kinase. Raf then phosphorylates and stimulates the kinase activity of MAPK kinase (MAPKK, MEK) on a key serine residue. MAPKK then phosphorylates and activates MAPK (ERK), which can phosphorylate a variety of cytoplasmic and membrane-bound substrates or, more importantly, translocate into the nucleus where it phosphorylates and activates specific transcription factors to effect novel gene expression.

Figure 8-4. Schematic structure of the ErbB2, ErbB3, and ErbB4 receptors. The receptors contain two cysteine-rich domains that are located extracellularly (ovals): a transmembrane span and a cytoplasmically located tyrosine kinase (rounded oblongs). Both heterodimerization and homodimerization can occur, and it appears that the various ligands listed have specificity for a given receptor type. HB-EGF, heparin-binding epidermal growth factor; NRG, neuregulin.

(From Garratt AN, et al. Trends Cardiovasc Med 2003;13:80, by permission.)Copyright © 2003
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