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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 -
Methods in Molecular Biology (Clifton,... 2023Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a...
Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a gene. The main purpose of cloning is to study either that DNA sequence or the RNA or protein product it encodes. Building on key enzymatic discoveries in the late 1960s, gene cloning was pioneered in the early 1970s. Since then, DNA cloning and manipulation have been used in every area of biological and biomedical research, from molecular genetics, structural biology, and developmental biology to neurobiology, ancient DNA studies, and immunology. It is a versatile technique that can be applied to a variety of starting DNA types and lengths, including cDNAs, genes, gene fragments, chromosomal regions, or shorter fragments such as PCR products and functional control regions such as enhancers or promoters. The starting DNA can originate from any cell, tissue, or organism. In this chapter we will cover traditional ("classic") molecular cloning strategy. This comprises six linked stages in which (1) PCR is used to amplify a DNA region of interest that is then (2) digested with restriction enzymes, alongside a selected vector, to produce complementary ends crucial for the two molecules to be (3) ligated by an ATP-dependent DNA ligase, creating a recombinant DNA molecule. The recombinant DNA is then (4) introduced into competent bacterial cells by transformation and (5) grown on a selective agar media, followed by (6) colony-PCR for screening purposes. We provide a worked example to demonstrate the cloning of an average-size gene (in this case the 2 kb DNA ligase A gene) from E. coli into a common plasmid expression vector. We have included six color figures and two tables to depict the key stages of a classical molecular cloning protocol. If you are cloning a segment of DNA or a gene, remember that each DNA cloning experiment is unique in terms of sequence, length, and experimental purpose. However, the principles of traditional cloning covered in this chapter are the same for any DNA sequence; we have included a detailed notes section, so you should easily be able to transfer them to your own work. Some of the following chapters in this volume will cover other, more recently developed, cloning protocols.
Topics: DNA, Recombinant; Escherichia coli; Cloning, Molecular; Polymerase Chain Reaction; Genetic Vectors; DNA Ligase ATP
PubMed: 36853452
DOI: 10.1007/978-1-0716-3004-4_1 -
Molecular Cell Jul 2023Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We...
Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay called insertion and deletion sequencing (Indel-seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentally induced and spontaneous genome instability. Templated insertions, which derive from sequences genome wide, require contact between donor and acceptor loci, require homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop, followed by DNA synthesis, displacement, and then ligation by non-homologous end joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that is distinct from cut-and-paste events.
Topics: Humans; DNA Repair; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA; Genomic Instability; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 37402370
DOI: 10.1016/j.molcel.2023.06.016 -
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 -
Cold Spring Harbor Protocols Dec 2019This protocol, suitable for both general and close-proximity mutagenesis, includes a simple and rapid procedure that combines polymerase chain reaction (PCR), DpnI...
This protocol, suitable for both general and close-proximity mutagenesis, includes a simple and rapid procedure that combines polymerase chain reaction (PCR), DpnI digestion, and overlap extension. The key point of this approach is the use of overlap extension to form a circular DNA plasmid with mutations without the need for phosphorylated primers or ligase reactions. Essentially, during the first round of PCR, the new DNA is synthesized with nicks between the 3' ends of the synthesized DNA and the 5' ends of the first pair of primers. During successive rounds of PCR, a new pair of mutagenic oligonucleotides leads to the synthesis of two DNA segments that anneal together with the overlap sequence inside the two primers. This new mutated molecule also contains nicks but at different positions compared with those formed in the first round of PCR that had been "repaired" by overlap extension. Mutations can be introduced successfully by this method. Finally, the circular DNA is transformed into cells, where the nicks are ligated into a circular plasmid. One important requirement is that the parental plasmid carrying the target gene needs to be methylated by Dam methyltransferase or purified from Dam (i.e., DH5a).
Topics: DNA; Mutagenesis, Site-Directed; Plasmids; Polymerase Chain Reaction
PubMed: 31792139
DOI: 10.1101/pdb.prot097816 -
Methods in Molecular Biology (Clifton,... 2022In situ HiC uses the relative frequency of DNA-DNA ligation events to reconstruct the three-dimensional architecture of a genome. As such, restriction enzyme digested...
In situ HiC uses the relative frequency of DNA-DNA ligation events to reconstruct the three-dimensional architecture of a genome. As such, restriction enzyme digested ends of genomic DNA within fixed nuclei are tagged with biotinylated dNTPs. DNA-DNA ligation events generated via proximity ligation are then captured, amplified and next generation sequenced to determine their linear genomic position, but also their three-dimensional relationship. Here, we describe these steps in detail.
Topics: Cell Nucleus; Chromatin; DNA; Genome; Genomics
PubMed: 35103976
DOI: 10.1007/978-1-0716-2140-0_18 -
Methods in Molecular Biology (Clifton,... 2021Ligation of a hairpin oligonucleotide to genomic DNA prior to bisulfite conversion and PCR amplification physically links the two complementary DNA strands. This...
Ligation of a hairpin oligonucleotide to genomic DNA prior to bisulfite conversion and PCR amplification physically links the two complementary DNA strands. This additional step in the conversion procedure overcomes the limitations of conventional bisulfite sequencing where information of the cytosine methylation status is only obtained from one of the two strands of an individual DNA molecule. Sequences derived from hairpin bisulfite PCR products reveal the dynamics of this epigenetic memory system on both strands of individual DNA molecules. The chapter describes a reliable step-by-step procedure to generate hairpin-linked DNA. It also provides a guide for efficient bisulfite conversion that is suitable for both conventional and hairpin bisulfite sequencing approaches.
Topics: Cytosine; DNA; DNA Methylation; DNA, Complementary; Humans; Inverted Repeat Sequences; Nucleic Acid Conformation; Oligonucleotides; Polymerase Chain Reaction; Sequence Analysis, DNA; Sulfites
PubMed: 32822039
DOI: 10.1007/978-1-0716-0876-0_22 -
Methods in Molecular Biology (Clifton,... 2021CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic...
CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic reactions, however the process becomes time-consuming as the number of sgRNAs increases and does not allow the incorporation of chemical modifications that can improve or expand the functionality of CRISPR. Solid-phase RNA synthesis can overcome these issues, but highly pure full-length sgRNA remains at the limits of current synthetic methods. Here, we demonstrate a "split-and-click" approach that separates the sgRNA into its two smaller components - a DNA-targeting ~20-mer RNA and a constant Cas9-binding 79-mer RNA - and chemically ligates them together to generate a biologically active sgRNA. The benefits of our approach lie in the stringent purification of the DNA-targeting 20-mer, the reduced synthesis of the constant 79-mer each time a new sgRNA is required, and the rapid access it provides to custom libraries of sgRNAs.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Gene Editing; Humans; RNA, Guide, CRISPR-Cas Systems
PubMed: 32926378
DOI: 10.1007/978-1-0716-0687-2_5 -
Molecular Cell Feb 2024Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA...
Nucleosomes, the basic structural units of chromatin, hinder recruitment and activity of various DNA repair proteins, necessitating modifications that enhance DNA accessibility. Poly(ADP-ribosyl)ation (PARylation) of proteins near damage sites is an essential initiation step in several DNA-repair pathways; however, its effects on nucleosome structural dynamics and organization are unclear. Using NMR, cryoelectron microscopy (cryo-EM), and biochemical assays, we show that PARylation enhances motions of the histone H3 tail and DNA, leaving the configuration of the core intact while also stimulating nuclease digestion and ligation of nicked nucleosomal DNA by LIG3. PARylation disrupted interactions between nucleosomes, preventing self-association. Addition of LIG3 and XRCC1 to PARylated nucleosomes generated condensates that selectively partition DNA repair-associated proteins in a PAR- and phosphorylation-dependent manner in vitro. Our results establish that PARylation influences nucleosomes across different length scales, extending from the atom-level motions of histone tails to the mesoscale formation of condensates with selective compositions.
Topics: Nucleosomes; Poly ADP Ribosylation; Poly(ADP-ribose) Polymerases; Cryoelectron Microscopy; Biomolecular Condensates; DNA Repair; Histones; DNA; DNA Damage; Poly (ADP-Ribose) Polymerase-1
PubMed: 38215753
DOI: 10.1016/j.molcel.2023.12.019