> Physiology > Molecular Function > DNA Cleavage

DNA Cleavage

DNA Cleavage is the process of breaking the phosphodiester bonds within the DNA molecule, often facilitated by enzymes or chemical agents.
This process is essential for various genetic engineering techniques, gene editing, and the study of DNA repair mechanisms.
Researchers leverage DNA cleavage to investigate genome structure, gene expression, and the impact of genetic variations.
Understanding the optimal conditions and methods for DNA cleavage can enhance the reproducibility, accuracy, and efficiency of these important research endeavors.

Most cited protocols related to «DNA Cleavage»

CRISPR Off-Target DNA Cleavage Assay

DNA cleavage assay was carried out based on [3 (link)] with commercially available Cas9 enzyme (NEB). PCR amplified genomic fragments for each sgRNA-1 off-target site (OT#1_F 5’-AGAGGCAAGTAAAGGTCAAGTAGG-3’, OT#1_R 5’-TCACATTGCAATGATGAGCACTTT-3’; OT#2_F 5’-CCAGCTCATGTTGAAAAGACACAT-3’, OT#2_R 5’-CCCCCACAGATGAAATGAAAAGAC-3’; OT#3_F 5’-TACCCAAAAATTGTAAGCCAGCAG-3’, OT#3_R 5’-AGATCTGATCCGGTTTCAAAGTGA-3’) were cloned into pGEM-T easy vector (Promega). The plasmids were pre-linearized with BsaI (NEB) about 2kb from the sgRNA target site. 3nM of the pre-linearized plasmids were incubated for one 1h with 30nM sgRNA-1 and 30nM Cas9 protein (NEB) and supplemented with Cas9 nuclease buffer (NEB) in a 30μl reaction volume at 37°C. Gel electrophoresis was performed on 1.5% agarose gel in 1x TAE buffer (40mM Tris, 20mM acetic acid, 1mM EDTA).

Stemmer M., Thumberger T., del Sol Keyer M., Wittbrodt J, & Mateo J.L. (2015). CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool. PLoS ONE, 10(4), e0124633.

Publication 2015

Acetic Acid Biological Assay Buffers Cloning Vectors CRISPR-Associated Protein 9 DNA Cleavage Edetic Acid Electrophoresis Genome Plasmids Promega prostaglandin M Sepharose tris-acetate-EDTA buffer Tromethamine

Quantitative Yeast Assay for TALEN Function

The yeast assay for TALEN function was adapted from one we developed previously for ZFNs (8 (link),24 (link)) in which cleavage of the target, positioned between partially duplicated fragments of the lacZ gene, reconstitutes the gene via subsequent recombination to provide a quantitative readout (Supplementary Figure S3a). For typical heterodimeric target sites (i.e. such as would typically occur in a native DNA sequence), paired TALEN constructs, in pTAL3 and pTAL4, are transformed together into yeast strain YPH500 (α mating type) using histidine and leucine prototrophy for selection. Individual TALEN monomers can be tested on homodimeric sites using just one of these plasmids. The target is made using synthesized complementary oligonucleotides that produce BglII- and SpeI-compatible ends and cloned between the lacZ fragments in the high copy DNA cleavage reporter plasmid pCP5 (24 (link)) cut with those enzymes (Supplementary Figure S3b). The target plasmid is transformed into yeast strain YPH499 (α mating type), using tryptophan prototrophy for selection, but also excluding uracil from the growth medium: in addition to the target cloning site, pCP5 carries also the URA3 gene between the lacZ fragments so that selection for URA3 ensures that the strain has not undergone spontaneous recombination (and loss of URA3) prior to the assay.
Three transformants each of YPH500 carrying the TALEN construct(s) and of YPH499 carrying the target plasmid are cultured overnight at 30°C, with rotary shaking at 800 rpm, in synthetic complete medium lacking histidine and/or leucine (TALENs) or tryptophan and uracil (target). TALEN and target transformants are next mated (three pairs) by combining 200–500 µl of the overnight cultures, adding 1 ml of YPD medium and incubating for 4–6 h at 30°C, shaking at 250–300 rpm. Cells are harvested by centrifguation, washed in 1 ml synthetic complete medium lacking histidine and/or leucine and tryptophan, but now containing uracil, then resuspended in 5 ml of that medium and incubated overnight again at 30°C, with shaking (800 rpm), to an OD₆₀₀ between 0.1 and 0.9. Cells are harvested by centrifugation, then resuspended and lysed using YeastBuster Protein Extraction Reagent (Novagen) according to the manufacturer's protocol for small cultures. A total of 100 µl of lysate is transferred to a microtiter well plate and β-galactosidase activity measured and normalized as previously described (24 (link)). For high-throughput, yeast may be cultured and mated (using a gas permeable seal) as well as lysed in 24-well blocks. We typically express activity relative to a Zif268 ZFN (24 (link)).

Cermak T., Doyle E.L., Christian M., Wang L., Zhang Y., Schmidt C., Baller J.A., Somia N.V., Bogdanove A.J, & Voytas D.F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 39(12), e82.

Publication 2011

beta-Galactosidase Biological Assay Cells Centrifugation Cytokinesis DNA Cleavage DNA Sequence Enzymes Histidine LacZ Genes Leucine Oligonucleotides Permeability Phocidae Plasmids Proteins Recombination, Genetic Strains Transcription Activator-Like Effector Nucleases Tryptophan Uracil Yeast, Dried

Cas9 Cleavage and Binding Assay

The plasmid DNA substrate used in Fig. 2b contained a λ2 target sequence cloned into the EcoRI and BamHI sites on pUC19. Oligoduplex DNA substrates (Extended Data Table 1) were 55-bp in length and were prepared by mixing together complementary synthetic oligonucleotides (Integrated DNA Technologies) in Hybridization Buffer, heating to 95 °C for 1–2 min followed by slow-cooling, and purifying on a 5% native polyacrylamide gel (0.5X TBE buffer with 5 mM MgCl₂) run at 4 °C. When assayed directly, DNA substrates were 5'-radiolabeled using [γ-^{32 (link)}P]-ATP (Promega) and T4 polynucleotide kinase (New England Biolabs) or 3'-radiolabeled using [α-^{32 (link)}P]-dATP (Promega) and terminal transferase (New England Biolabs) (Extended Data Fig. 4). The substrates presented in Fig. 4b were prepared by 5'-radiolabeling only the target strand, hybridizing it to a 10X excess of the indicated unlabeled complementary strand, and gel purifying the partial/full duplex by 10% native gel electrophoresis.
Cas9:RNA complexes were reconstituted prior to cleavage and binding experiments by incubating Cas9 and the crRNA:tracrRNA duplex for 10 min at 37 °C in Reaction Buffer. Binding experiments used dCas9 (except as indicated in Extended Data Fig. 4) and either equimolar crRNA:tracrRNA or a 10X molar excess of crRNA:tracrRNA over dCas9 (Extended Data Fig. 3 & 8). Binding reactions contained 0.1–1 nM DNA and increasing apo-dCas9 or dCas9:RNA concentrations, and were incubated at 37 °C for one hour before being resolved by 5% native polyacrylamide gel electrophoresis (0.5X TBE buffer with 5 mM MgCl₂) run at 4 °C. DNA was visualized by phosphorimaging, quantified with ImageQuant (GE Healthcare), and analyzed with Kaleidagraph (Synergy Software).
Cleavage assays were conducted in Reaction Buffer at room temperature and analyzed by 1% agarose gel electrophoresis and ethidium bromide staining (Fig. 2b) or 10% denaturing polyacrylamide gel electrophoresis and phosphorimaging. Aliquots were removed at each time point and quenched by the addition of gel loading buffer supplemented with 25 mM EDTA (at 1X). Reactions contained ~1 nM radiolabeled DNA substrate and 10 nM Cas9:RNA (competition experiments, Fig. 3b,c,e, Extended Data Fig. 6) or 100 nM Cas9:RNA (Fig. 4a,b, Extended Data Fig. 1, 7, 8, 9). Competition experiments used λ1 target DNA and were supplemented with 500 nM unlabeled competitor DNAs (Fig. 3c,e) or an extended concentration range of competitor DNAs (Extended Data Fig. 6). All oligoduplex DNA cleavage experiments were visualized by phosphorimaging and quantified with ImageQuant (GE Healthcare)

Sternberg S.H., Redding S., Jinek M., Greene E.C, & Doudna J.A. (2014). DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507(7490), 62-67.

Publication 2014

Biological Assay Buffers Crossbreeding crRNA, Transactivating Cytokinesis Deoxyribonuclease EcoRI DNA DNA Cleavage Edetic Acid Electrophoresis Electrophoresis, Agar Gel Ethidium Bromide Magnesium Chloride Molar Native Polyacrylamide Gel Electrophoresis Oligonucleotides Plasmids Polyacrylamide Gel Electrophoresis polyacrylamide gels Polynucleotide 5'-Hydroxyl-Kinase Promega RNA, CRISPR Guide Transferase Tris-borate-EDTA buffer

Generating Recombinant Membrane Proteins

The codon optimized zebrafish DHHC15 (zfDHHC15) sequence was cloned into a modified version of pPICZ-C vector with an His10 tag followed by a GFP coding sequence and finally, by PreScission cleavage site at the N terminus of the zfDHHC15 encoding DNA sequence. Further, site directed mutagenesis was performed to mutate Cys153 to serine to get the zfDHHS15 expression construct. The optimized vector harboring zfDHHC15 or zfDHHS15 gene was transformed into Pichia pastoris HIS + Cells SDM1163 cells. Pichia were transformed using standard methods and the transformants were selected on YPDS plates containing 400–800 μg/mL zeocin.
The human DHHC20 (hDHHC20) expression construct was made similarly with N-terminal His10 tag, GFP and PreScission cleavage sequences without any modification/truncation of the hDHHC20 sequence and was transformed into Pichia following the same protocol.
A vector containing the sequence for human Snap25b was obtained from Addgene (Plasmid #53235) and the gene was cloned into pET28-Prx by digestion/ligation using NdeI/BamHI. The original TEV cleavage site was mutated to a PreScission cleavage site.

Rana M.S., Kumar P., Lee C.J., Verardi R., Rajashankar K.R, & Banerjee A. (2018). Fatty acyl recognition and transfer by an integral membrane S-acyltransferase. Science (New York, N.Y.), 359(6372), eaao6326.

Publication 2018

Cells Cloning Vectors Codon Cytokinesis Digestion DNA Cleavage FLJ25952 protein, human Genes Homo sapiens Komagataella pastoris Ligation Mutagenesis, Site-Directed Open Reading Frames Pichia Plasmids Serine Zebrafish Zeocin

DNA Cleavage Assay for Bacterial Topoisomerase IV

DNA cleavage reactions were carried out using the procedure of Fortune and Osheroff (41 (link)). Reactions contained 200 nM wild-type or mutant topoisomerase IV and 10 nM negatively supercoiled pBR322 in a total of 20 μL of cleavage buffer [40 mM Tris–HCl (pH 7.9), 10 mM MgCl₂, 50 mM NaCl, and 2.5% (v/v) glycerol]. In some reactions, the concentration dependence of MgCl₂ was examined or the divalent metal ion was replaced with either CaCl₂ or MnCl₂. Reaction mixtures were incubated at 37 °C for 10 min, and enzyme-DNA cleavage complexes were trapped by the addition of 2 μL of 5% SDS followed by 1 μL of 250 mM EDTA (pH 8.0). Proteinase K (2 μL of a 0.8 mg/mL solution) was added, and samples were incubated at 45 °C for 45 min to digest the enzyme. Samples were mixed with 2 μL of agarose gel loading buffer, heated at 45 °C for 5 min, and subjected to electrophoresis in 1% agarose gels in 40 mM Tris-acetate (pH 8.3) and 2 mM EDTA containing 0.5 μg/mL ethidium bromide. DNA bands were visualized and quantified as described above. DNA cleavage was monitored by the conversion of supercoiled plasmid to linear molecules.
Assays that monitored the DNA cleavage activities of wild-type and mutant B. anthracis topoisomerase IV in the absence of drugs substituted 1 mM CaCl₂ for 10 mM MgCl₂ in the cleavage buffer. Assays that assessed the DNA cleavage activities of the wild-type and mutant enzymes in the presence of drugs contained 0-30 μM compound for the wild-type enzyme and 0-500 μM compound for the mutant enzymes.
For assays that monitored competition between ciprofloxacin (0-150 μM) and 8-methyl-quinazoline-2,4-dione (20 μM), the level of cleavage seen with the corresponding concentration of ciprofloxacin in the absence of the quinazolinedione was used as a baseline and was subtracted from the cleavage level seen in the presence of both compounds. Ciprofloxacin and 8-methyl-quinazoline-2,4-dione were added simultaneously to reaction mixtures.

Aldred K.J., McPherson S.A., Wang P., Kerns R.J., Graves D.E., Turnbough CL J.r, & Osheroff N. (2011). Drug Interactions with Bacillus anthracis Topoisomerase IV: Biochemical Basis for Quinolone Action and Resistance. Biochemistry, 51(1), 370-381.

Publication 2011

Acetate Bacillus anthracis Biological Assay Buffers Ciprofloxacin compound 30 Cytokinesis DNA Cleavage DNA Topoisomerase IV Edetic Acid Electrophoresis Endopeptidase K Enzymes Ethidium Bromide Gels Glycerin Magnesium Chloride manganese chloride Metals Multienzyme Complexes Pharmaceutical Preparations Plasmids Quinazolinediones Quinazolines Sepharose Sodium Chloride Tromethamine Vision

Most recents protocols related to «DNA Cleavage»

CasX CRISPR Plasmid Interference in E. coli

Not available on PMC !

Example 2

FIGS. 4A-4C. Plasmid Interference by CasX expressed in E. coli. Experimental design of CasX plasmid interference. Competent E. coli cells expressing the minimal interference CasX locus (acquisition proteins removed) were prepared. These cells were transformed with a plasmid containing a match to the spacer in the CasX CRISPR locus (target) or not (non-target) and plated on media containing antibiotic selection for the CRISPR and target plasmid. Successful plasmid interference results in reduced number of transformed colonies for the target plasmid. FIG. 4B cfu/ug of transformed plasmid containing spacer from CasX1 (sX1), spacer from CasX2 (sX2) or a non-target plasmid containing a random 30 nt sequence. FIG. 4C serial dilution was performed of transformants from FIG. 4B on media containing antibiotic selection for both the CRISPR and target plasmid.

FIGS. 5A-5B PAM dependent plasmid interference by CasX. PAM depletion assays were conducted with CasX. E. coli containing the CasX CRISPR locus were transformed with a plasmid library with 7 nucleotides randomized 5′ or 3′ of the target sequence. The target plasmid was selected for and transformants were pooled. The randomized region was amplified and prepared for deep sequencing. Depleted sequences were identified and used to generate a PAM logo. FIG. 5B PAM logo generated for deltaproteobacteria CasX showed a strong preference for sequences containing a 5′-TTCN-3′ flanking sequence 5′ of the target. A 3′ PAM was not detected. c, PAM logo generated for planctomyces CasX showed a strong preference for sequences containing a 5′-TTCN-3′ flanking sequence 5′ of the target with lower stringency at the first T. A 3′ PAM was not detected.

FIGS. 6A-6C. CasX is a dual-guided CRISPR-Cas effector complex. FIG. 6A CRISPR locus for tracrRNA knockout experiments and sgRNA tests. FIG. 6B colony forming units (cfu) per g of transformed plasmid containing a target or non-target sequence. Deletion of the tracrRNA resulted in ablation of plasmid interference. Expression of a synthetic sgRNA in place of the tracrRNA and CRISPR array resulted in robust plasmid interference by CasX. FIG. 6C diagram of sgRNA design (derived from tracrRNA and crRNA sequences for CasX1). The tracrRNA (green) was joined to the crRNA (repeat, black; spacer, red) by a tetraloop (GAAA).

FIG. 7. Schematic of CasX RNA guided DNA interference. CasX binds to a tracrRNA (green) and the crRNA (black, repeat; red, spacer). Base pairing of the guide RNA to the target sequence (blue) containing the correct protospacer adjacent motif (yellow) results in double stranded cleavage of the target DNA. The depicted sequences are derived from tracrRNA and crRNA sequences for CasX1.

US11873504B2. RNA-guided nucleic acid modifying enzymes and methods of use thereof (2024-01-16). The Regents of the University of California [US]. Inventors: Jennifer A Doudna [US], Jillian F. Banfield [US], David Burstein [US], Lucas Benjamin Harrington [US], Steven C. Strutt [US].

Patent 2024

Antibiotics Biological Assay Cells Clustered Regularly Interspaced Short Palindromic Repeats CRISPR Loci crRNA, Transactivating Deletion Mutation Deltaproteobacteria DNA Cleavage DNA Library Enzymes Escherichia coli Nucleic Acids Nucleotides Plasmids Proteins RNA, CRISPR Guide Technique, Dilution

Stability of DNA Nanostructures in Physiological and Acidic Environments

In the physiological environment: The working solution for each DNA nanostructure was adjusted to 20 nM and then mixed with DMEM (supplemented with 20% FBS) at a 1:1 volume ratio. All samples were immediately incubated at 37°C for 2, 4, and 6 h.
In the low-pH environment: To mimic the cleavage situation of DNA nanostructures in cellular lysosomes, DOS, DTU, and DTE were treated in the low-pH environment (pH = 5) for different periods at 37°C.
Right after incubation, different DNA samples were subjected to 10% native polyacrylamide gel electrophoresis (PAGE) at 100 V for 1 h (gel prepared in 1 × TBE buffer supplemented with 10 mM MgCl₂). After the electrophoresis, the gels were carefully recovered and incubated with 1 h in the staining solution (3 × SYBR Safe was dissolved in 1 × TBE buffer). Finally, the stained gels were exposed using the Tanon 4600SF multifunctional imaging system (Tanon, China).

Liu F., Liu X., Gao W., Zhao L., Huang Q, & Arai T. (2023). Transmembrane capability of DNA origami sheet enhanced by 3D configurational changes. iScience, 26(3), 106208.

Publication 2023

Cells DNA Cleavage Electrophoresis Gels Lysosomes Magnesium Chloride Native Polyacrylamide Gel Electrophoresis physiology Tris-borate-EDTA buffer

Quantification of Apoptotic Retinal Cells via TUNEL Assay

In situ cell death detection kit (Roche) based on labeling of DNA strand breaks [(TdT-mediated dUTP-X nick end labeling (TUNEL)] was used to quantify apoptotic retinal cells (Table 1, Additional file 1: Table S1). Briefly, the DNA cleavage can be detected by labeling the free 3′-OH termini with fluorescein modified nucleotides in an enzymatic reaction. According to the manufacturer’s protocol, retinal cell death detection was conducted using 14 µm retinal cryosections from 4% PFA (w/v) fixed eyes (methods described before). After three washes with PB, slides were incubated in phosphate buffered saline (PBS) with 1% Triton X-100 (v/v) for 5–10 min at RT in humidity chamber. Then, TUNEL mix reagent was incubated for 1 h at 37 °C in dark conditions. Thereafter, the reaction was stopped with three PB washes for 5 min at RT in the dark. Finally, preparations were mounted using Citifluor and coverslipped.

Albertos-Arranz H., Martínez-Gil N., Sánchez-Sáez X., Noailles A., Monferrer Adsuara C., Remolí Sargues L., Pérez-Santonja J.J., Lax P., Calvo Andrés R, & Cuenca N. (2023). Microglia activation and neuronal alterations in retinas from COVID-19 patients: correlation with clinical parameters. Eye and Vision, 10, 12.

Publication 2023

Apoptosis Cell Death Cells Cryoultramicrotomy deoxyuridine triphosphate DNA Breaks DNA Cleavage Enzymes Eye Fluorescein Humidity In Situ Nick-End Labeling Nucleotides Phosphates Retina Saline Solution Triton X-100

CRISPR-Cas9 Mediated Knockout of Sporulation Genes

Knockout mutants were constructed by partially exchanging the coding regions of the cotH genes to a functional pyrG gene (CBS277.49. v2.0 genome database ID Mucci1.e_gw1.3.865.1), which complements the uracil auxotrophy of the applied strain. This gene replacement was carried out by homology-driven repair (HDR) following a CRISPR-Cas9 strategy described previously (29 (link), 61 (link)). Protospacer sequences designed to target the DNA cleavage in the cotH1, cotH2, cotH3, cotH4, and cotH5 genes are presented in Table S2A. Using these sequences, Alt-R CRISPR RNA (crRNA) and Alt-R CRISPR-Cas9 transactivating crRNA (tracrRNA) molecules were designed and purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA). To form the crRNA:tracrRNA duplexes (i.e., the guide RNAs [gRNAs]), IDT nuclease-free duplex buffer (IDT, Coralville, IA, USA) was used according to the instructions of the manufacturer. Deletion cassettes functioning also as the template DNAs for the HDR were constructed by PCR using the Phusion Flash high-fidelity PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA). First, two fragments upstream and downstream from the protospacer sequence of the corresponding cotH gene and the entire pyrG gene along with its promoter and terminator sequences were amplified using the primers listed in Table S1B. The amplified fragments were fused in a subsequent PCR using nested primers (Table S1B) where the ratio of concentrations of the fragments was 1:1:1. For each transformation procedure, 5 μg template DNA, 10 μM gRNA, and 10 μM Cas9 nuclease enzyme (Alt-R S.p. Cas9 nuclease; IDT, Coralville, IA, USA) were introduced together into the M. lusitanicus MS12 strain by polyethylene glycol (PEG)-mediated protoplast transformation (29 (link), 62 (link)). Potential mutant colonies were selected on solid YNB medium by complementing the uracil auxotrophy of the MS12 strain. From each primary transformant, monosporangial colonies were formed under selective conditions. Disruption of the cotH genes and the presence of the integrated pyrG gene were proven by PCR using the primers listed in Table S1B and sequencing of the amplified fragment. Sequencing and PCR revealed that the CRISPR-Cas9-mediated HDR caused the expected modification (i.e., disruption of the cotH genes by the integration of the pyrG) in the targeted sites. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis indicated the lack of cotH transcripts in all transformants.

Szebenyi C., Gu Y., Gebremariam T., Kocsubé S., Kiss-Vetráb S., Jáger O., Patai R., Spisák K., Sinka R., Binder U., Homa M., Vágvölgyi C., Ibrahim A.S., Nagy G, & Papp T. (2023). cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus. mBio, 14(1), e03386-22.

Publication 2023

Buffers Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Associated Protein 9 crRNA, Transactivating Deletion Mutation DNA Cleavage Genes Genome Oligonucleotide Primers Polyethylene Glycols Protoplasts Real-Time Polymerase Chain Reaction RNA Strains Terminator Regions, Genetic Transcription, Genetic Uracil

CRISPR/Cas9 and TALEN Genomic Cleavage Detection

For the detection of genomic DNA cleavage by CRISPR/Cas9 and TALEN approaches, we used the GeneArt Genomic Cleavage Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, hiPSCs were transfected with sgRNA or TALEN, and four days later, PCR amplification of the desired locus was performed and run in a gel to ensure a single band. Next, the PCR product was subjected to several rounds of denaturation and re-annealing to generate mismatches, which were detected and cleaved by using the Detection Enzyme. The results were visualized by gel electrophoresis with iBrightCL1000 (Thermo Fisher Scientific, Waltham, MA, USA) and band intensity quantification was performed with iBright^TM Analysis Software (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Band intensity quantification was correlated with the Cas9 or TALEN activity.

Siles L., Gaudó P, & Pomares E. (2023). High-Efficiency CRISPR/Cas9-Mediated Correction of a Homozygous Mutation in Achromatopsia-Patient-Derived iPSCs. International Journal of Molecular Sciences, 24(4), 3655.

Publication 2023

Clustered Regularly Interspaced Short Palindromic Repeats Cytokinesis DNA Cleavage Electrophoresis Enzymes Genome Human Induced Pluripotent Stem Cells Transcription Activator-Like Effector Nucleases

Top products related to «DNA Cleavage»

Geneart genomic cleavage detection kit by Thermo Fisher Scientific

Sourced in United States

The GeneArt Genomic Cleavage Detection Kit is a laboratory tool designed for the detection and analysis of genomic cleavage events. It provides a reliable method for identifying and quantifying gene editing outcomes in cell lines and samples.

In situ cell death detection kit by Roche

Sourced in Germany, United States, Switzerland, China, United Kingdom, France, Canada, Belgium, Japan, Italy, Spain, Hungary, Australia

The In Situ Cell Death Detection Kit is a laboratory product designed for the detection of programmed cell death, or apoptosis, in cell samples. The kit utilizes a terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks, allowing for the visualization and quantification of cell death. The core function of this product is to provide researchers with a tool to study and analyze cell death processes.

Hiseq x ten sequencer by Illumina

Sourced in United States, China

The HiSeq X Ten sequencer is a high-throughput DNA sequencing system designed and manufactured by Illumina. It is capable of generating large amounts of genomic data by simultaneously processing multiple samples. The core function of the HiSeq X Ten is to perform DNA sequencing, which is the process of determining the precise order of nucleotides within a DNA molecule.

Proteinase k by New England Biolabs

Sourced in United States, United Kingdom, France, Australia, Germany, Macao

Proteinase K is a broad-spectrum serine protease that hydrolyzes proteins. It is commonly used in molecular biology and biochemistry applications to degrade proteins, such as in the isolation of nucleic acids from biological samples.

Dneasy tissue kit by Qiagen

Sourced in Germany, United States, Spain, France, Canada, China, United Kingdom, Japan, Switzerland, Netherlands, Italy

The DNeasy Tissue Kit is a DNA extraction and purification system designed for the isolation of genomic DNA from a variety of sample types, including animal tissues, plant materials, and microorganisms. The kit utilizes a spin-column-based procedure to efficiently capture and purify DNA, which can then be used in downstream applications such as PCR, sequencing, and other molecular biology techniques.

Rna loading dye by New England Biolabs

Sourced in United States

2× RNA loading dye is a buffer solution used to prepare RNA samples for gel electrophoresis. It facilitates the loading of RNA samples onto agarose or polyacrylamide gels and helps track the progress of the electrophoresis run.

Sybr safe by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Australia, Canada, France, Germany, China, Denmark

SYBR Safe is a sensitive nucleic acid stain used for detecting DNA or RNA in agarose gels. It exhibits low toxicity and can be used in place of traditional ethidium bromide staining.

End repair mix by Thermo Fisher Scientific

Sourced in United States

End Repair Mix is a laboratory reagent used to repair the ends of DNA fragments in preparation for further processing, such as ligation or sequencing. It functions by converting the 3' overhangs and 5' overhangs of DNA into blunt ends, enabling downstream applications.

Cutsmart buffer by New England Biolabs

Sourced in United States, China, United Kingdom

CutSmart buffer is a universal buffer designed for use with a variety of restriction enzymes. It maintains optimal enzyme activity and provides consistent, reliable DNA cleavage.

Rnase a by Qiagen

Sourced in Germany, United States, Australia, United Kingdom, Netherlands, Switzerland, Japan, Spain, Canada

RNase A is a laboratory enzyme used for the digestion and degradation of RNA. It functions by catalyzing the hydrolysis of single-stranded RNA molecules, breaking down the phosphodiester bonds between the ribonucleotides.

What are the different types or variations of DNA Cleavage?

DNA Cleavage can occur through various mechanisms, including enzymatic cleavage by restriction enzymes, chemical cleavage using agents like hydroxyl radicals or endonucleases, and physical cleavage via techniques like sonication or mechanical shearing. Each method has its own advantages and limitations, depending on the specific research application and desired outcome.

How can DNA Cleavage be used in genetic engineering and gene editing?

DNA Cleavage is a fundamental process in genetic engineering and gene editing techniques. Researchers leverage cleavage to insert, remove, or modify specific DNA sequences, enabling the creation of transgenic organisms, gene knockouts, and precise genome editing. Understanding the optimal conditions for DNA cleavage is crucial for ensuring the efficiency, accuracy, and reproducibility of these important genetic manipulation procedures.

What are some common challenges or considerations when working with DNA Cleavage?

Some key challenges in DNA Cleavage include ensuring complete and specific cleavage, minimizing off-target effects, and maintaining the integrity of the DNA fragments. Factors like enzyme specificity, reaction conditions, and the complexity of the target DNA sequence can all impact the effectiveness and reliability of the cleavage process. Careful optimization and method validation are essential to overcome these hurdles and achieve consistent, high-quality results.

How can PubCompare.ai help researchers optimize their DNA Cleavage protocols?

PubCompare.ai's AI-driven platform can greatly assist researchers in optimizing their DNA Cleavage protocols. The tool allows you to efficiently screen the literature and patents to idenfity the most effective and reproducible methods for your specific research needs. The platform's AI analysis can highlight key differences in protocol effectiveness, enzme specificity, and other critical factors, enabling you to choose the best approach for your work and improve the accuracy and efficiency of your DNA Cleavage experiments.

What are some of the key applications of DNA Cleavage in scientific research?

DNA Cleavage has a wide range of applications in scientific research, including: 1. Gene editing and genome engineering: Leveraging cleavage to modify or insert specific DNA sequences. 2. Genetic analysis: Studying DNA fragmentation patterns to investigate genome structure and gene expression. 3. DNA repair mechanisms: Analyzing the cellular response to DNA breaks and the pathways involved in repairing cleavage sites. 4. Epigenetic studies: Using cleavage to investigate DNA methylation and chromatin remodeling. 5. Diagnostic and forensic applications: Utilizing cleavage patterns for genetic profiling and identification purposes.

More about "DNA Cleavage"

DNA cleavage, also known as DNA breakage or DNA fragmentation, is a critical process in various genetic engineering and biotechnology applications.
This process involves the disruption of the phosphodiester bonds within the DNA molecule, often facilitated by enzymes or chemical agents.
Understanding the optimal conditions and methods for DNA cleavage is essential for enhancing the reproducibility, accuracy, and efficiency of research endeavors.
Researchers leverage DNA cleavage to investigate genome structure, gene expression, and the impact of genetic variations.
Techniques like the GeneArt Genomic Cleavage Detection Kit and the In Situ Cell Death Detection Kit employ DNA cleavage to analyze genome editing and cell death, respectively.
The HiSeq X Ten sequencer, a high-throughput DNA sequencing platform, also relies on DNA cleavage during library preparation.
Enzymes like Proteinase K and chemical agents like the DNeasy Tissue Kit and 2× RNA loading dye are commonly used to facilitate DNA cleavage.
The SYBR Safe dye is often used to visualize and quantify cleaved DNA fragments.
End Repair Mix and CutSmart buffer are also important components in DNA cleavage procedures, ensuring optimal enzyme activity and precision.
By understanding the various aspects of DNA cleavage, researchers can enhance their ability to study genome structure, gene expression, and the impact of genetic variations.
This knowledge can lead to advancements in genetic engineering, gene editing, and DNA repair mechanisms, ultimately driving progress in numerous fields of science and medicine.