-
BioEssays : News and Reviews in... Nov 1993A number of novel gene detection techniques all revolve around the ligation of synthetic nucleic acid probes. In such ligase-assisted gene detection reactions, specific... (Review)
Review
A number of novel gene detection techniques all revolve around the ligation of synthetic nucleic acid probes. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of probe molecules. The probes are designed to hybridize immediately next to each other on the target nucleic acid strand. Demonstration of ligated probes results in highly specific detection of and efficient distinction between similar sequence variants under standard reaction conditions. Accordingly, the principle has been applied in automated genetic screening procedures. Ligation reactions are also integral to a number of amplification procedures and they will be of value in an expanding range of genetic analyses.
Topics: DNA; DNA Ligases; Genetic Techniques; Models, Structural; Nucleic Acid Conformation; Oligonucleotide Probes
PubMed: 8292008
DOI: 10.1002/bies.950151112 -
Methods (San Diego, Calif.) Nov 2012We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. This method is based on Chromosome Conformation Capture, in which...
We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. This method is based on Chromosome Conformation Capture, in which chromatin is crosslinked with formaldehyde, then digested, and re-ligated in such a way that only DNA fragments that are covalently linked together form ligation products. The ligation products contain the information of not only where they originated from in the genomic sequence but also where they reside, physically, in the 3D organization of the genome. In Hi-C, a biotin-labeled nucleotide is incorporated at the ligation junction, enabling selective purification of chimeric DNA ligation junctions followed by deep sequencing. The compatibility of Hi-C with next generation sequencing platforms makes it possible to detect chromatin interactions on an unprecedented scale. This advance gives Hi-C the power to both explore the biophysical properties of chromatin as well as the implications of chromatin structure for the biological functions of the nucleus. A massively parallel survey of chromatin interaction provides the previously missing dimension of spatial context to other genomic studies. This spatial context will provide a new perspective to studies of chromatin and its role in genome regulation in normal conditions and in disease.
Topics: Animals; Cells, Cultured; Chromatin; Chromosome Mapping; Cross-Linking Reagents; DNA; DNA Fragmentation; Epistasis, Genetic; Fixatives; Formaldehyde; Gene Library; Genome, Human; Humans; Nucleic Acid Conformation; Sequence Analysis, DNA; Tissue Fixation
PubMed: 22652625
DOI: 10.1016/j.ymeth.2012.05.001 -
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 -
Bioconjugate Chemistry Mar 2020We report a chemical DNA-DNA ligation method based on copper-catalyzed azide-alkyne cycloaddition (CuAAC). We demonstrate that ion addition dramatically influences the...
We report a chemical DNA-DNA ligation method based on copper-catalyzed azide-alkyne cycloaddition (CuAAC). We demonstrate that ion addition dramatically influences the efficiency of the so-called click reaction. Even without any further additions, such as typically splint oligonucleotides for preorganization, the "click ligation" yields up to ∼83% product without any byproducts. Additionally, purification of the desired product is straightforward. In comparison to enzymatic ligation methods used to introduce adapters into, e.g., mRNA library preparation, this targeted chemical ligation method exhibits several advantages: increased ligated product and no adapter or cDNA oligomers byproducts. The advantages of the click ligation method were demonstrated by incorporation of azide modified nucleotides by several enzymes as well as broad polymerase acceptance of the obtained triazole linkage in PCR.
Topics: Alkynes; Azides; Catalysis; Click Chemistry; Copper; Cycloaddition Reaction; DNA; Models, Molecular; Nucleic Acid Conformation
PubMed: 31874033
DOI: 10.1021/acs.bioconjchem.9b00805 -
Nucleic Acids Research Aug 2021The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures...
The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.
Topics: DNA; DNA Ligases; Electrophoresis, Agar Gel; Ethidium; Kinetics; Microscopy, Atomic Force; Nanostructures; Nucleic Acid Conformation; Nucleic Acid Denaturation; Phosphorylation; Temperature; Thermodynamics
PubMed: 34289063
DOI: 10.1093/nar/gkab611 -
Nucleic Acids Research Feb 2014Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of...
Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.
Topics: DNA; DNA Ligases; Kinetics; RNA; Viral Proteins
PubMed: 24203707
DOI: 10.1093/nar/gkt1032 -
Chembiochem : a European Journal of... Nov 2016Coumarin moieties react with thymine and cytosine in DNA by photoinduced [2+2] cycloaddition, which allows quantitative DNA interstrand crosslink (ICL) formation. Here,...
Coumarin moieties react with thymine and cytosine in DNA by photoinduced [2+2] cycloaddition, which allows quantitative DNA interstrand crosslink (ICL) formation. Here, we report the application of coumarin analogues for DNA photoligation and the rearrangement of coumarin-induced ligation to ICL products. Both DNA sequences and the linker units at position 4 of the coumarin moieties affected coumarin-induced DNA photoligation. A flexible linker unit favored DNA ICL formation but led to inefficient photoligation, whereas coumarins without linker units greatly increased DNA photoligation efficiency. DNA photoligation induced by the coumarin moiety was photoswitchable. Ligation products were formed between coumarin and dT or dC upon 350 nm irradiation but reverted to the original single-stranded oligodeoxyribonucleotides (ODNs) upon 254 nm irradiation. Rearrangement of ligated ODNs into ICL products occurred during the switchable (350 nm/254 nm) processes. Additionally, photoinduced cleavage of coumarin 3 occurred with dC-3 cycloadducts upon 254 nm irradiation, which was confirmed by mass spectrometry analysis.
Topics: Coumarins; Cross-Linking Reagents; DNA; Humans; Molecular Structure; Photochemical Processes
PubMed: 27558701
DOI: 10.1002/cbic.201600240 -
Chemical Record (New York, N.Y.) 2001A systematic approach evaluating template-directed ligation reactions has now resulted in a simple outline for a two-stage replication cycle. This cycle builds on an... (Review)
Review
A systematic approach evaluating template-directed ligation reactions has now resulted in a simple outline for a two-stage replication cycle. This cycle builds on an efficient method for reading the information encoded in DNA into an amplified translation product. It is further demonstrated that the translation product strand is capable of catalyzing the synthesis of the original DNA strand. We propose that this cycle represents just one of many possible solutions; other chemical ligation or polymerization reactions could be accommodated with different templates. In that context, a new template, derived by modest changes to the DNA backbone, has been developed and has been shown to hybridize under reaction conditions different than those accessible to DNA. Therefore, the conceptual groundwork has been laid for extending this approach to encoding and reading stored information in molecules other than the natural biopolymers at the densities found in biology.
Topics: Combinatorial Chemistry Techniques; DNA; Oligonucleotides; Templates, Genetic; Transcription, Genetic
PubMed: 11893058
DOI: 10.1002/1528-0691(2001)1:1<53::AID-TCR8>3.0.CO;2-N -
Mutation Research Jul 1993Many carcinogens and mutagens interact with DNA to form specific adducts. Base-specificity and sequence-specificity of adduct formation has been analyzed previously with... (Review)
Review
Many carcinogens and mutagens interact with DNA to form specific adducts. Base-specificity and sequence-specificity of adduct formation has been analyzed previously with cloned, end-labelled DNA fragments. However, the distribution of adducts along a mammalian chromosome may be modulated by chromatin structure and could be different from that in naked plasmid DNA. Recently, a method has been developed that utilizes the sensitivity of the polymerase chain reaction (PCR) to detect DNA adducts at the DNA sequence level in mammalian cells. The sequence position of adducts can be mapped whenever it is possible to convert the adduct, either chemically or enzymatically, into a DNA strand break with a 5'-phosphate group. Fragments containing these ligatable breaks are amplified in a single-sided, ligation-mediated PCR reaction. We have used ligation-mediated PCR for detection of alkylguanine adducts and UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. We discuss the sensitivity of the method, its limitations, and its potential for mapping other DNA adducts at the DNA sequence level in mammalian cells.
Topics: Animals; DNA; DNA Damage; DNA Mutational Analysis; Humans; Ligands; Mammals; Polymerase Chain Reaction; Restriction Mapping; Ultraviolet Rays
PubMed: 7686264
DOI: 10.1016/0027-5107(93)90206-u -
Biochemistry Oct 2004The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction...
The DNA ligation reaction of topoisomerase II is essential for genomic integrity. However, it has been impossible to examine many fundamental aspects of this reaction because ligation assays historically required the enzyme to cleave a DNA substrate before sealing the nucleic acid break. Recently, a cleavage-independent DNA ligation assay was developed for human topoisomerase IIalpha [Bromberg, K. D., Hendricks, C., Burgin, A. B., and Osheroff, N. (2002) J. Biol. Chem. 277, 31201-31206]. This assay overcomes the requirement for DNA cleavage by monitoring the ability of the enzyme to ligate a nicked oligonucleotide in which the 5'-terminal phosphate at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. The cleavage-independent ligation assay was used to more fully characterize the DNA ligation activity of human topoisomerase IIalpha. Results suggest that the active site tyrosine contributes little to the catalysis of DNA ligation beyond its primary role as an activating/leaving group. Although arginine 804 (the residue immediately N-terminal to the active site tyrosine) has been proposed to help anchor the 5'-DNA terminus during cleavage, conversion of this residue to alanine had only a modest effect on DNA ligation. Thus, it appears that arginine 804 does not play an essential role in DNA strand joining. In contrast, disruption of base pairing at the 5'-DNA terminus abrogated DNA ligation in the absence of a covalent enzyme-DNA bond. Therefore, it is proposed that base pairing represents a secondary mechanism for aligning the 5'-DNA termini for ligation. Finally, the human enzyme appears to ligate the two scissile bonds of a cleavage site in a nonconcerted fashion.
Topics: Antigens, Neoplasm; Base Pairing; Catalysis; DNA Damage; DNA Repair; DNA Topoisomerases, Type II; DNA, Superhelical; DNA-Binding Proteins; Humans; Hydrolysis; Oligonucleotides; Plasmids; Substrate Specificity
PubMed: 15491148
DOI: 10.1021/bi049420h