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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 -
Nature Nov 2021Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different...
Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response.
Topics: Animals; Apoptosis; Cells, Cultured; Chromosome Mapping; DNA Fragmentation; DNA Ligase ATP; DNA, Circular; Endodeoxyribonucleases; Gene Expression Regulation; Genome; Immunity, Innate; Male; Membrane Proteins; Mice; Poly-ADP-Ribose Binding Proteins
PubMed: 34671165
DOI: 10.1038/s41586-021-04009-w -
Cell Jul 2018Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation....
Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.
Topics: Cell Nucleolus; Cell Nucleus; Chromosome Mapping; Chromosomes; DNA; Eukaryota; Genome; Humans; Structure-Activity Relationship
PubMed: 29887377
DOI: 10.1016/j.cell.2018.05.024 -
Autophagy Jan 2024AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E:...
AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E: hematoxylin and eosin staining; LCN2/NGAL: lipocalin 2; LPS: lipopolysaccharide; LTL: lotus tetragonolobus lectin; mKeima: mitochondria-targeted Keima; mtDNA: mitochondrial DNA; PAS: periodic acid - Schiff staining; RTECs: renal tubular epithelial cells; SAKI: sepsis-induced acute kidney injury; Scr: serum creatinine; SIRT3: sirtuin 3; TFAM: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine.
Topics: Humans; Sirtuin 3; Melatonin; Mitophagy; Autophagy; Acute Kidney Injury; Lipopolysaccharides; DNA, Mitochondrial; Sepsis; Kidney; DNA-Binding Proteins; Transcription Factors; Mitochondrial Proteins
PubMed: 37651673
DOI: 10.1080/15548627.2023.2252265 -
Nucleic Acids Research Jun 2017DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly...
DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.
Topics: Animals; Bone and Bones; DNA; DNA Ligases; DNA Primers; DNA, Single-Stranded; Fossils; Gene Library; High-Throughput Nucleotide Sequencing; Horses; Humans; Liver; Nucleic Acid Hybridization; Oligonucleotides; Polymerase Chain Reaction; Sequence Analysis, DNA; Swine
PubMed: 28119419
DOI: 10.1093/nar/gkx033 -
Genome Research Mar 2023Tn5 transposon tagments double-stranded DNA and RNA/DNA hybrids to generate nucleic acids that are ready to be amplified for high-throughput sequencing. The nucleic acid...
Tn5 transposon tagments double-stranded DNA and RNA/DNA hybrids to generate nucleic acids that are ready to be amplified for high-throughput sequencing. The nucleic acid substrates for the Tn5 transposon must be explored to increase the applications of Tn5. Here, we found that the Tn5 transposon can transpose oligos into the 5' end of single-stranded DNA longer than 140 nucleotides. Based on this property of Tn5, we developed a tagmentation-based and ligation-enabled single-stranded DNA sequencing method called TABLE-seq. Through a series of reaction temperature, time, and enzyme concentration tests, we applied TABLE-seq to strand-specific RNA sequencing, starting with as little as 30 pg of total RNA. Moreover, compared with traditional dUTP-based strand-specific RNA sequencing, this method detects more genes, has a higher strand specificity, and shows more evenly distributed reads across genes. Together, our results provide insights into the properties of Tn5 transposons and expand the applications of Tn5 in cutting-edge sequencing techniques.
Topics: DNA, Single-Stranded; DNA; Base Sequence; RNA; Sequence Analysis, RNA; DNA Transposable Elements
PubMed: 36958795
DOI: 10.1101/gr.277213.122 -
Cell Nov 2022Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution....
Methods for acquiring spatially resolved omics data from complex tissues use barcoded DNA arrays of low- to sub-micrometer features to achieve single-cell resolution. However, fabricating such arrays (randomly assembled beads, DNA nanoballs, or clusters) requires sequencing barcodes in each array, limiting cost-effectiveness and throughput. Here, we describe a vastly scalable stamping method to fabricate polony gels, arrays of ∼1-micrometer clonal DNA clusters bearing unique barcodes. By enabling repeatable enzymatic replication of barcode-patterned gels, this method, compared with the sequencing-dependent array fabrication, reduced cost by at least 35-fold and time to approximately 7 h. The gel stamping was implemented with a simple robotic arm and off-the-shelf reagents. We leveraged the resolution and RNA capture efficiency of polony gels to develop Pixel-seq, a single-cell spatial transcriptomic assay, and applied it to map the mouse parabrachial nucleus and analyze changes in neuropathic pain-regulated transcriptomes and cell-cell communication after nerve ligation.
Topics: Mice; Animals; Transcriptome; Chronic Pain; DNA; RNA; Gels
PubMed: 36368323
DOI: 10.1016/j.cell.2022.10.021 -
Biosensors & Bioelectronics Aug 2022In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free...
In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.
Topics: Animals; Biosensing Techniques; CRISPR-Cas Systems; DNA; Mice; Nucleic Acid Amplification Techniques; RNA; RNA, Messenger
PubMed: 35430408
DOI: 10.1016/j.bios.2022.114256 -
Nature Communications Jul 2018ChIP-seq and ChIP-exo identify where proteins bind along any genome in vivo. Although ChIP-seq is widely adopted in academic research, it has inherently high noise. In...
ChIP-seq and ChIP-exo identify where proteins bind along any genome in vivo. Although ChIP-seq is widely adopted in academic research, it has inherently high noise. In contrast, ChIP-exo has relatively low noise and achieves near-base pair resolution. Consequently, and unlike other genomic assays, ChIP-exo provides structural information on genome-wide binding proteins. Construction of ChIP-exo libraries is technically difficult. Here we describe greatly simplified ChIP-exo methods, each with use-specific advantages. This is achieved through assay optimization and use of Tn5 tagmentation and/or single-stranded DNA ligation. Greater library yields, lower processing time, and lower costs are achieved. In comparing assays, we reveal substantial limitations in other ChIP-based assays. Importantly, the new ChIP-exo assays allow high-resolution detection of some protein-DNA interactions in organs and in as few as 27,000 cells. It is suitable for high-throughput parallelization. The simplicity of ChIP-exo now makes it a highly appropriate substitute for ChIP-seq, and for broader adoption.
Topics: Animals; Binding Sites; Brain; Catalysis; Chromatin Immunoprecipitation; DNA; DNA, Single-Stranded; DNA-Binding Proteins; Gene Library; Genomics; Humans; K562 Cells; Kidney; Liver; Lung; Mice; Protein Binding; Saccharomyces cerevisiae; Sequence Analysis, DNA; Transcription Factors
PubMed: 30030442
DOI: 10.1038/s41467-018-05265-7 -
International Journal of Molecular... Aug 2021species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of... (Review)
Review
species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T-DNA often contains, at its junctions with plant DNA, deletions of T-DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T-DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T-DNA integration. However, the involvement of specific plant DNA repair proteins and proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T-DNA integration into the plant genome.
Topics: Agrobacterium; Chromosomes, Plant; DNA Repair; DNA, Bacterial; DNA, Plant; Plants; Transformation, Genetic
PubMed: 34445162
DOI: 10.3390/ijms22168458