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Molekuliarnaia Biologiia 2016"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful... (Review)
Review
"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.
Topics: Cloning, Molecular; DNA, Recombinant; Genetic Engineering; Genetic Vectors
PubMed: 27028806
DOI: 10.7868/S0026898416010134 -
Journal of Biosciences Jun 2019Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the molecular biology of DNA. Forty years ago, the... (Review)
Review
Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the molecular biology of DNA. Forty years ago, the scientists whose pioneering work had explored the activity and sequence specificity of these enzymes, contributing to the definition of their enormous potential as tools for DNA characterization, mapping and manipulation, were awarded the Nobel Prize. In this short review, we celebrate the history of these enzymes in the light of their many different uses, as these proteins have accompanied the history of DNA for over 50 years representing active witnesses of major steps in the field.
Topics: CRISPR-Cas Systems; Chromatin; Chromosome Mapping; Cloning, Molecular; DNA; DNA Methylation; DNA Restriction Enzymes; History, 20th Century; History, 21st Century; Humans; Molecular Biology; Nobel Prize; Nucleotide Mapping; Transcription Activator-Like Effector Nucleases
PubMed: 31180051
DOI: No ID Found -
Current Protocols Jul 2021The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a...
The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a chromosome conformation capture (3C)-based technology to detect pair-wise chromatin interactions genome-wide, and has become a benchmark tool to study genome organization. In Hi-C, chromatin conformation is first captured by chemical cross-linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high-throughput sequencing, which enables the mapping of pair-wise chromatin interactions genome-wide. Over the past decade, "next-generation" sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in-depth guide to performing an up-to-date Hi-C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross-linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi-C procedure that enables the detection of genome folding features at a wide range of distances. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fixation of nuclear conformation Basic Protocol 2: Chromosome conformation capture Basic Protocol 3: Hi-C sequencing library preparation.
Topics: Animals; Chromatin; Chromosomes; Genome; High-Throughput Nucleotide Sequencing; Nucleic Acid Conformation
PubMed: 34286910
DOI: 10.1002/cpz1.198 -
Nature Microbiology Oct 2022The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including...
The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species.
Topics: Antiviral Agents; Bacteriophages; CRISPR-Cas Systems; Escherichia coli; Prophages
PubMed: 36123438
DOI: 10.1038/s41564-022-01219-4 -
Nature Protocols Jun 2019R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear...
R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.
Topics: Animals; Antibodies; Antibodies, Monoclonal; DNA; High-Throughput Nucleotide Sequencing; Humans; Immunoprecipitation; Mice; Nucleic Acid Conformation; Nucleic Acid Hybridization; Polymerase Chain Reaction; RNA
PubMed: 31053798
DOI: 10.1038/s41596-019-0159-1 -
Journal of the American Statistical... 2022Numerical embedding has become one standard technique for processing and analyzing unstructured data that cannot be expressed in a predefined fashion. It stores the main...
Numerical embedding has become one standard technique for processing and analyzing unstructured data that cannot be expressed in a predefined fashion. It stores the main characteristics of data by mapping it onto a numerical vector. An embedding is often unsupervised and constructed by transfer learning from large-scale unannotated data. Given an embedding, a downstream learning method, referred to as a two-stage method, is applicable to unstructured data. In this article, we introduce a novel framework of embedding learning to deliver a higher learning accuracy than the two-stage method while identifying an optimal learning-adaptive embedding. In particular, we propose a concept of -minimal sufficient learning-adaptive embeddings, based on which we seek an optimal one to maximize the learning accuracy subject to an embedding constraint. Moreover, when specializing the general framework to classification, we derive a graph embedding classifier based on a hyperlink tensor representing multiple hypergraphs, directed or undirected, characterizing multi-way relations of unstructured data. Numerically, we design algorithms based on blockwise coordinate descent and projected gradient descent to implement linear and feed-forward neural network classifiers, respectively. Theoretically, we establish a learning theory to quantify the generalization error of the proposed method. Moreover, we show, in linear regression, that the one-hot encoder is more preferable among two-stage methods, yet its dimension restriction hinders its predictive performance. For a graph embedding classifier, the generalization error matches up to the standard fast rate or the parametric rate for linear or nonlinear classification. Finally, we demonstrate the utility of the classifiers on two benchmarks in grammatical classification and sentiment analysis. Supplementary materials for this article are available online.
PubMed: 36936129
DOI: 10.1080/01621459.2020.1775614 -
PloS One 2021In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a...
In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a laborious manual process, Bionano Genomics platforms now offer high-throughput, automated optical mapping based on chips packed with nanochannels through which unwound DNA is guided and the fluorescent DNA backbone and specific restriction sites are recorded. Although the raw image data obtained is of high quality, the processing and assembly software accompanying the platforms is closed source and does not seem to make full use of data, labeling approximately half of the measured signals as unusable. Here we introduce two new software tools, independent of Bionano Genomics software, to extract and process molecules from raw images (OptiScan) and to perform molecule-to-molecule and molecule-to-reference alignments using a novel signal-based approach (OptiMap). We demonstrate that the molecules detected by OptiScan can yield better assemblies, and that the approach taken by OptiMap results in higher use of molecules from the raw data. These tools lay the foundation for a suite of open-source methods to process and analyze high-throughput optical mapping data. The Python implementations of the OptiTools are publicly available through http://www.bif.wur.nl/.
Topics: Chromosome Mapping; Genomics; High-Throughput Nucleotide Sequencing; Optical Restriction Mapping; Sequence Analysis, DNA
PubMed: 34591846
DOI: 10.1371/journal.pone.0253102 -
GigaScience 2015Optical Mapping is an established single-molecule, whole-genome analysis system, which has been used to gain a comprehensive understanding of genomic structure and to... (Review)
Review
Optical Mapping is an established single-molecule, whole-genome analysis system, which has been used to gain a comprehensive understanding of genomic structure and to study structural variation of complex genomes. A critical component of Optical Mapping system is the image processing module, which extracts single molecule restriction maps from image datasets of immobilized, restriction digested and fluorescently stained large DNA molecules. In this review, we describe robust and efficient image processing techniques to process these massive datasets and extract accurate restriction maps in the presence of noise, ambiguity and confounding artifacts. We also highlight a few applications of the Optical Mapping system.
Topics: Animals; Chromosome Mapping; Genome; Humans; Image Processing, Computer-Assisted; Sensitivity and Specificity
PubMed: 26617984
DOI: 10.1186/s13742-015-0096-z -
Ultrasound in Obstetrics & Gynecology :... Jul 2022Doppler techniques are needed for the evaluation of the intraplacental circulation and can be of great value in the diagnosis of placental anomalies. Highly sensitive... (Review)
Review
Doppler techniques are needed for the evaluation of the intraplacental circulation and can be of great value in the diagnosis of placental anomalies. Highly sensitive Doppler techniques can differentiate between the maternal (spiral arteries) and fetal (intraplacental branches of the umbilical artery) components of the placental circulation and assist in the evaluation of the placental functional units. A reduced number of placental functional units can be associated with obstetric complications, such as fetal growth restriction. Doppler techniques can also provide information on decidual vessels and blood movement. Abnormal decidual circulation increases the risk of placenta accreta. Doppler evaluation of the placenta greatly contributes to the diagnosis and clinical management of placenta accreta, vasa previa, placental infarcts, placental infarction hematoma, maternal floor infarction, massive perivillous fibrin deposition and placental tumors. However, it has a limited role in the diagnosis and clinical management of placental abruption, placental hematomas, placental mesenchymal dysplasia and mapping of placental anastomoses in monochorionic twin pregnancies. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.
Topics: Female; Fetal Growth Retardation; Humans; Infarction; Placenta; Placenta Accreta; Placenta Diseases; Pregnancy; Ultrasonography, Doppler; Ultrasonography, Prenatal
PubMed: 34806234
DOI: 10.1002/uog.24816 -
AIDS (London, England) Sep 2015HIV type 1 (HIV-1) has a very narrow host range that is limited to humans and chimpanzees. HIV-1 cannot replicate well in Old World monkey cells such as rhesus and... (Review)
Review
HIV type 1 (HIV-1) has a very narrow host range that is limited to humans and chimpanzees. HIV-1 cannot replicate well in Old World monkey cells such as rhesus and cynomolgus monkeys. Tripartite motif (TRIM)5α is a key molecule that confers potent resistance against HIV-1 infection and is composed of really interesting new gene, B-box2, coiled-coil and PRYSPRY domains. Interaction between TRIM5α PRYSPRY domains and HIV-1 capsid core triggers the anti-HIV-1 activity of TRIM5α. Analysis of natural HIV variants and extensive mutational experiments has revealed the presence of critical amino acid residues in both the PRYSPRY domain and HIV capsid for potent HIV suppression by TRIM5α. Genetic manipulation of the human TRIM5 gene could establish human cells totally resistant to HIV-1, which may lead to a cure for HIV-1 infection in the future.
Topics: Animals; Antiviral Restriction Factors; Carrier Proteins; HIV Core Protein p24; HIV-1; Humans; Macaca fascicularis; Macaca mulatta; Protein Binding; Protein Interaction Mapping; Protein Structure, Tertiary; Proteins; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 26372380
DOI: 10.1097/QAD.0000000000000812