Sequence replication can occur during different steps of the sequencing protocol, and can therefore generate artificial duplicates (Gomez-Alvarez et al., 2009 (link)). Here, duplicates are categorized into the following groups: (i) exact duplicate, (ii) 5′ duplicate (sequence matches the 5′-end of a longer sequence), (iii) 3′ duplicate, (iv) exact duplicate with the reverse complement of another sequence and (v) 5′/3′ duplicate with the reverse complement of another sequence. The duplicates are identified independently by sorting and prefix/suffix matching of the sequences.
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Physiology
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Genetic Function
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DNA Replication
DNA Replication
DNA replication is the fundamental biological process by which a DNA molecule is duplicated, producing two identical copies.
This essential process ensures the accurate transmission of genetic information from one cell generation to the next.
During replication, the double-stranded DNA molecule is unwound and separated, serving as a template for the synthesis of new complementary strands.
A complex of enzymes and proteins, including DNA polymerase, primase, and helicase, orchestrate this intricate process.
Understanding DNA replication is crucial for advancing research in areas such as genetics, cell biology, and molecular biology.
Optimizing DNA replication protocols can drive forward innovation in fields like DNA sequencing, gene editing, and synthetic biology.
Expereince the future of DNA replication optimization today with PubCompare.ai, the AI-driven platform revolutionizing this critical area of study.
This essential process ensures the accurate transmission of genetic information from one cell generation to the next.
During replication, the double-stranded DNA molecule is unwound and separated, serving as a template for the synthesis of new complementary strands.
A complex of enzymes and proteins, including DNA polymerase, primase, and helicase, orchestrate this intricate process.
Understanding DNA replication is crucial for advancing research in areas such as genetics, cell biology, and molecular biology.
Optimizing DNA replication protocols can drive forward innovation in fields like DNA sequencing, gene editing, and synthetic biology.
Expereince the future of DNA replication optimization today with PubCompare.ai, the AI-driven platform revolutionizing this critical area of study.
Most cited protocols related to «DNA Replication»
All samples were obtained under institutional IRB approval and with documented informed consent. A complete list of samples is given in Table S2 . Whole-exome capture libraries were constructed and sequenced on Illumina HiSeq flowcells to average coverage of 118x. Whole-genome sequencing was done with the Illumina GA-II or Illumina HiSeq sequencer, achieving an average of ~30X coverage depth. Reads were aligned to the reference human genome build hg19 using an implementation of the Burrows-Wheeler Aligner, and a BAM file was produced for each tumor and normal sample using the Picard pipeline6 (link). The Firehose pipeline was used to manage input and output files and submit analyses for execution. The MuTect30 and Indelocator (Sivachenko, A. et al., manuscript in preparation) algorithms were used to identify somatic single-nucleotide variants (SSNVs) and short somatic insertions and deletions, respectively. Mutation spectra were analyzed using non-negative matrix factorization (NMF). Significantly mutated genes were identified using MutSigCV, which estimates the background mutation rate (BMR) for each gene-patient-category combination based on the observed silent mutations in the gene and noncoding mutations in the surrounding regions. Because in most cases these data are too sparse to obtain accurate estimates, we increased accuracy by pooling data from other genes with similar properties (e.g. replication time, expression level). Significance levels (p-values) were determined by testing whether the observed mutations in a gene significantly exceed the expected counts based on the background model. False Discovery Rates (q-values) were then calculated, and genes with q≤0.1 were reported as significantly mutated. Full methods details are listed in Supplementary Information .
Diploid Cell
DNA Replication
Exome
Gene Deletion
Genes
Genes, vif
Genetic Background
Genome, Human
Insertion Mutation
Multiple Acyl Coenzyme A Dehydrogenase Deficiency
Mutation
Neoplasms
Nucleotides
Patients
Silent Mutation
Total RNA was extracted from EBV transformed lymphoblastoid cell line pellets by the TRIzol reagent (Ambion), and mRNA and small RNA sequencing of 465 unique individuals was performed on the Illumina HiSeq2000 platform, with paired-end 75bp mRNA-seq and single-end 36bp small RNA-seq. Five samples were sequenced in replicate in each of the seven sequencing laboratories. The mRNA and small RNA reads were mapped with GEM31 and miraligner32 (link), respectively, with an average of 48.9M mRNA-seq reads and 1.2M miRNA reads per sample after QC. Numerous transcript features were quantified using Gencode v1233 (link) and miRBase v1834 (link) annotations: protein-coding and lincRNA genes (16,084 detected in >50% of samples), transcripts (67,603; with FluxCapacitor7 (link)), exons (146,498), annotated splice junctions (129,805; analyzed in detail in Ferreira et al. submitted), transcribed repetitive elements (47,409), and mature miRNAs (715). Data quality was assessed by sample correlations and read and gene count distributions, and technical variation was removed by PEER normalization35 (link) for the QTL and miRNA-mRNA correlation analyses11 (link). The samples clustered uniformly both before and after normalization. The genotype data was obtained from 1000 Genomes Phase 1 data set for 421 samples (80× average exome and 5× whole genome read depth), and the remaining 41 samples were imputed from Omni 2.5M SNP array data. Furthermore, we did functional reannotation for all the 1000 Genomes variants using Gencode v12. QTL mapping was done with linear regression, using genetic variants with >5% frequency in 1MB window and normalized quantifications transformed to standard normal. Permutations were used to adjust FDR to 5%. Full details are provided in Supplementary Methods .
Cell Line, Transformed
DNA Replication
Exome
Exons
Genes
Genetic Diversity
Genome
Genotype
Long Intergenic Non-Protein Coding RNA
MicroRNAs
Pellets, Drug
Proteins
Repetitive Region
RNA, Messenger
RNA-Seq
trizol
A main limitation of efficiency calibrated method and ΔΔCt method is that only one set of cDNA samples are employed to determine the amplification efficiency. It was assumed that the same amplification efficiency could be applied to other cDNA samples as long as the primers and amplification conditions are the same. However, amplification efficiency not only depends on the primer characteristics, but also varies among different cDNA samples. Using a standard curve for only one set of tested samples to derive the amplification efficiency might overlook the error introduced by sample differences. In our experimental design, we have performed standard curve experiments with four concentrations of three replicates for all samples and genes involved. The ΔΔCt will derive from the standard curves only, and the data quality is examined for each gene and sample combination. The analysis of two samples is presented in the paper as an example. A minimal of PCRs of two replicates in three concentrations will be required for each sample. Even though more effort is required, the data is more reliable out of stringent data quality control and data analysis based on statistical models.
The output dataset included Ct number, gene name, sample name, concentration and replicate. We used Microsoft® Excel to open the exported Ct file from an ABI 7000 sequence analysis system and then to transform data into a tab delimited text file for SAS processing. The sample data set is shown in Table1 .
All programs were developed with SAS 9.1 (SAS Institute).
The output dataset included Ct number, gene name, sample name, concentration and replicate. We used Microsoft® Excel to open the exported Ct file from an ABI 7000 sequence analysis system and then to transform data into a tab delimited text file for SAS processing. The sample data set is shown in Table
All programs were developed with SAS 9.1 (SAS Institute).
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DNA, Complementary
DNA Replication
Genes
Oligonucleotide Primers
Polymerase Chain Reaction
ChIP-seq analysis was performed in biological replicate as described4 using antibodies validated by Western blots and peptide competitions. ChIP DNA and input controls were sequenced using the Illumina Genome Analyzer. Expression profiles were acquired using Affymetrix GeneChip arrays. Chromatin states were learned jointly by applying an HMM8 to 10 data tracks for each of the 9 cell types. We focused on a 15 state model that provides sufficient resolution to resolve biologically-meaningful patterns yet is reproducible across cell types when independently processed. We used this model to produce 9 genome-wide chromatin state annotations, which were validated by additional ChIP experiments and reporter assays. Multi-cell type clustering was conducted on locations assigned to strong promoter state 1 (or strong enhancer state 4) in at least one cell type using the k-means algorithm. Enhancer-target gene linkages were predicted by correlating normalized signal intensities of H3K27ac, H3K4me1 and H3K4me2 with gene expression across cell types as a function of distance to the TSS. Upstream regulators were predicted using a set of known TF motifs assembled from multiple sources. Motif instances were identified by sequence match and evolutionary conservation. P-values for GWAS studies were based on randomizing the location of SNPs, and the FDR based on randomizing the assignment of SNPs across studies. Datasets are available from the ENCODE website (http://genome.ucsc.edu/ENCODE ), the supporting website for this paper (http://compbio.mit.edu/ENCODE_chromatin_states ), and the Gene Expression Omnibus (GSE26386).
Antibodies
Biological Assay
Biological Evolution
Biopharmaceuticals
Cells
Chromatin
Chromatin Immunoprecipitation Sequencing
DNA Chips
DNA Replication
Gene Chips
Gene Expression
Genome
Genome-Wide Association Study
Linkage, Genetic
Peptides
Physiology, Cell
Single Nucleotide Polymorphism
Western Blot
Most recents protocols related to «DNA Replication»
Example 7
The MTT Cell Proliferation assay determines cell survival following apple stem cell extract treatment. The purpose was to evaluate the potential anti-tumor activity of apple stem cell extracts as well as to evaluate the dose-dependent cell cytotoxicity.
Principle: Treated cells are exposed to 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). MTT enters living cells and passes into the mitochondria where it is reduced by mitochondrial succinate dehydrogenase to an insoluble, colored (dark purple) formazan product. The cells are then solubilized with DMSO and the released, solubilized formazan is measured spectrophotometrically. The MTT assay measures cell viability based on the generation of reducing equivalents. Reduction of MTT only occurs in metabolically active cells, so the level of activity is a measure of the viability of the cells. The percentage cell viability is calculated against untreated cells.
Method: A549 and NCI-H520 lung cancer cell lines and L132 lung epithelial cell line were used to determine the plant stem cell treatment tumor-specific cytotoxicity. The cell lines were maintained in Minimal Essential Media supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a 5% CO2 at 37 Celsius. Cells were seeded at 5×103 cells/well in 96-well plates and incubated for 48 hours. Triplicates of eight concentrations of the apple stem cell extract were added to the media and cells were incubated for 24 hours. This was followed by removal of media and subsequent washing with the phosphate saline solution. Cell proliferation was measured using the MTT Cell Proliferation Kit I (Boehringer Mannheim, Indianapolis, IN) New medium containing 50 μl of MTT solution (5 mg/ml) was added to each well and cultures were incubated a further 4 hours. Following this incubation, DMSO was added and the cell viability was determined by the absorbance at 570 nm by a microplate reader.
In order to determine the effectiveness of apple stem cell extracts as an anti-tumor biological agent, an MTT assay was carried out and IC50 values were calculated. IC50 is the half maximal inhibitory function concentration of a drug or compound required to inhibit a biological process. The measured process is cell death.
Results: ASC-Treated Human Lung Adenocarcinoma Cell Line A549.
Results: ASC-Treated Human Squamous Carcinoma Cell Line NCI-H520.
Results: ASC-treated Lung Epithelial Cell Line L132.
Summary Results: Cytotoxicity of Apple Stem Cell Extracts.
Apple stem cell extracts killed lung cancer cells lines A549 and NCI-H520 at relatively low doses: IC50s were 12.58 and 10.21 μg/ml respectively as compared to 127.46 μg/ml for the lung epithelial cell line L132. Near complete anti-tumor activity was seen at a dose of 250 μg/ml in both the lung cancer cell lines. This same dose spared more than one half of the L132 cells. See Tables 7-10. The data revealed that apple stem cell extract is cytotoxic to lung cancer cells while sparing lung epithelial cells.
Example 9
The experiment of Example 7 was repeated substituting other plant materials for ASC. Plant stem cell materials included Dandelion Root Extract (DRE), Aloe Vera Juice (AVJ), Apple Fiber Powder (AFP), Ginkgo Leaf Extract (GLE), Lingonberry Stem Cells (LSC), Orchid Stem Cells (OSC) as described in Examples 1 and 2. The concentrations of plant materials used were nominally 250, 100, 50, 25, 6.25, 3.125, 1.562, and 0.781 μg/mL. These materials were tested only for cells the human lung epithelial cell line L132 (as a proxy for normal epithelial cells) and for cells of the human lung adenocarcinoma cell line A549 (as a proxy for lung cancer cells).
A549 cells lung cancer cell line cytotoxicity results for each of the treatment materials.
DRE-Treated Lung Cancer Cell Line A549 Cells.
AVJ-Treated Lung Cancer Cell line A549 Cells.
AFP-Treated Lung Cancer Cell line A549 Cells.
GLE-treated Lung Cancer Cell line A549 Cells.
LSC-treated lung cancer cell lines A549 cells.
OSC-treated Lung Cancer Cell line A549 Cells.
L132 cells (“normal” lung epithelial cell line) cytotoxicity results for each of the treatment materials.
DRE-Treated Lung Epithelial Cell Line L132 cells.
AVJ-Treated Lung Epithelial Cell Line L132 cells.
AFP-Treated Lung Epithelial Cell Line L132 cells.
GLE-Treated Lung Epithelial Cell Line L132 cells.
LSC-Treated Lung Epithelial Cell Line L132 cells.
OSC-Treated Lung Epithelial Cell Line L132 cells.
Calculated values.
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14-3-3 Proteins
43-63
61-26
A549 Cells
Action Potentials
Adenocarcinoma of Lung
Aloe
Aloe vera
Antineoplastic Agents
Biological Assay
Biological Factors
Biological Processes
Bromides
Cardiac Arrest
Cell Death
Cell Extracts
Cell Lines
Cell Proliferation
Cells
Cell Survival
Cytotoxin
diphenyl
DNA Replication
Epistropheus
Epithelial Cells
Fibrosis
Formazans
Genetic Selection
Ginkgo biloba
Ginkgo biloba extract
Homo sapiens
Lingonberry
Lung
Lung Cancer
Lung Neoplasms
Malignant Neoplasms
Mitochondria
Mitochondrial Inheritance
Neoplasms
Neoplastic Stem Cells
Oral Cavity
PEG SD-01
Penicillins
Pharmaceutical Preparations
Phosphates
Plant Cells
Plant Leaves
Plant Roots
Plants
Powder
Psychological Inhibition
Saline Solution
SD 31
SD 62
SEM-76
Squamous Cell Carcinoma
Stem, Plant
Stem Cells
Streptomycin
Succinate Dehydrogenase
Sulfoxide, Dimethyl
Taraxacum
Tetrazolium Salts
Example 1
Cells were treated with the indicated compounds for 7 hours before lysis with Buffer RLT (QIAGEN, Hilden, Germany) containing 1% β-mercaptoethanol. Total RNA was isolated using the Rneasy Plus Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. First-strand cDNA was synthesized using the SuperScript VILO Master Mix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Real-time qPCR was run on ViiA 7 Real Time PCR System (Thermo Fisher Scientific). For qRT-PCR, the expression of the reference gene glucuronidase beta (GUSB) was used to normalize expression of the target genes DUSP6, SPRY4, and glycogen synthase kinase 3 beta (GSK3B). Replicate qRT-PCR reactions were analyzed for each sample, and QuantStudio Real-Time PCR software (Life Technologies, Carlsbad, CA) normalized the average expression of DUSP6, SPRY4, or GSK3B to the average expression of the reference gene GUSB in each sample.
Full text: Click here
2-Mercaptoethanol
beta-Glucuronidase
Buffers
cabozantinib
Cells
DNA, Complementary
DNA Replication
DUSP6 protein, human
Gene Expression
Genes
Glycogen Synthase Kinase 3 beta
KIF5B protein, human
Neoplasms
Non-Small Cell Lung Carcinoma
Real-Time Polymerase Chain Reaction
Example 2
A. Seed Treatment with Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the isolated microbe as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
B. Seed Treatment with Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the microbial consortium as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
C. Treatment with Agricultural Composition Comprising Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
D. Treatment with Agricultural Composition Comprising Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
A. Seed Treatment with Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the isolated microbe as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
B. Seed Treatment with Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the microbial consortium as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
C. Treatment with Agricultural Composition Comprising Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the with the agricultural composition will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
D. Treatment with Agricultural Composition Comprising Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the with the agricultural composition will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
A. Seed Treatment with Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the isolated microbe as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
B. Seed Treatment with Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the microbial consortium as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
C. Treatment with Agricultural Composition Comprising Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
D. Treatment with Agricultural Composition Comprising Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
The inoculants were prepared from isolates grown as spread plates on R2A incubated at 25° C. for 48 to 72 hours. Colonies were harvested by blending with sterile distilled water (SDW) which was then transferred into sterile containers. Serial dilutions of the harvested cells were plated and incubated at 25° C. for 24 hours to estimate the number of colony forming units (CFU) in each suspension. Dilutions were prepared using individual isolates or blends of isolates (consortia) to deliver 1×105 cfu/microbe/seed and seeds inoculated by either imbibition in the liquid suspension or by overtreatment with 5% vegetable gum and oil.
Seeds corresponding to the plants of table 15 were planted within 24 to 48 hours of treatment in agricultural soil, potting media or inert growing media. Plants were grown in small pots (28 mL to 200 mL) in either a controlled environment or in a greenhouse. Chamber photoperiod was set to 16 hours for all experiments on all species. Air temperature was typically maintained between 22-24° C.
Unless otherwise stated, all plants were watered with tap water 2 to 3 times weekly. Growth conditions were varied according to the trait of interest and included manipulation of applied fertilizer, watering regime and salt stress as follows:
-
- Low N—seeds planted in soil potting media or inert growing media with no applied N fertilizer
- Moderate N—seeds planted in soil or growing media supplemented with commercial N fertilizer to equivalent of 135 kg/ha applied N
- Insol P—seeds planted in potting media or inert growth substrate and watered with quarter strength Pikovskaya's liquid medium containing tri-calcium phosphate as the only form phosphate fertilizer.
- Cold Stress—seeds planted in soil, potting media or inert growing media and incubated at 10° C. for one week before being transferred to the plant growth room.
- Salt stress—seeds planted in soil, potting media or inert growing media and watered with a solution containing between 100 to 200 mg/L NaCl.
Untreated (no applied microbe) controls were prepared for each experiment. Plants were randomized on trays throughout the growth environment. Between 10 and 30 replicate plants were prepared for each treatment in each experiment. Phenotypes were measured during early vegetative growth, typically before the V3 developmental stage and between 3 and 6 weeks after sowing. Foliage was cut and weighed. Roots were washed, blotted dry and weighed. Results indicate performance of treatments against the untreated control.
The data presented in table 15 describes the efficacy with which a microbial species or strain can change a phenotype of interest relative to a control run in the same experiment. Phenotypes measured were shoot fresh weight and root fresh weight for plants growing either in the absence of presence of a stress (assay). For each microbe species, an overall efficacy score indicates the percentage of times a strain of that species increased a both shoot and root fresh weight in independent evaluations. For each species, the specifics of each independent assay is given, providing a strain ID (strain) and the crop species the assay was performed on (crop). For each independent assay the percentage increase in shoot and root fresh weight over the controls is given.
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Ammonia
Asparagine
Aspartic Acid
Biological Assay
Bosea thiooxidans
Calcium Phosphates
Capsicum
Cells
Chlorophyll
Cold Shock Stress
Cold Temperature
Crop, Avian
Dietary Fiber
DNA Replication
Droughts
Drought Tolerance
Embryophyta
Environment, Controlled
Farmers
Fertilization
Glutamic Acid
Glutamine
Glycine
Growth Disorders
Herbaspirillum
Herbaspirillum huttiense
Leucine
Lolium
Lycopersicon esculentum
Lysine
Maize
Massilia niastensis
Methionine
Microbial Consortia
Nitrates
Nitrites
Nitrogen
Novosphingobium rosa
Paenibacillus
Paenibacillus amylolyticus
Pantoea agglomerans
Pantoea vagans
Phenotype
Phosphates
Photosynthesis
Plant Development
Plant Embryos
Plant Leaves
Plant Proteins
Plant Roots
Plants
Polaromonas ginsengisoli
Pseudoduganella violaceinigra
Pseudomonas
Pseudomonas fluorescens
Rahnella
Rahnella aquatilis
Retention (Psychology)
Rhodococcus erythropolis
Rosa
Salt Stress
Sodium Chloride
Sodium Chloride, Dietary
Stenotrophomonas chelatiphaga
Stenotrophomonas maltophilia
Stenotrophomonas rhizophila
Stenotrophomonas terrae
Sterility, Reproductive
Strains
Technique, Dilution
Threonine
Triticum aestivum
Tryptophan
Tyrosine
Vegetables
Zea mays
Example 3
Investigation of Virus Infectivity as a Factor that Determines Plaque Size.
With the revelation that plaque formation is strongly influenced by the immunogenicity of the virus, the possibility that infectivity of the virus could be another factor that determines plaque sizes was investigated. The uptake of viruses into cells in vitro was determined by measuring the amounts of specific viral RNA sequences through real-time PCR.
To measure total viral RNA, total cellular RNA was extracted using the RNEasy Mini kit (Qiagen), and complementary DNA synthesized using the iScript cDNA Synthesis kit (Bio-Rad). To measure total viral RNA, quantitative real-time PCR was done using a primer pair targeting a highly conserved region of the 3′ UTR common to all four serotypes of dengue; inter-sample normalization was done using GAPDH as a control. Primer sequences are listed in Table 5. Pronase (Roche) was used at a concentration of 1 mg/mL and incubated with infected cells for five minutes on ice, before washing with ice cold PBS. Total cellular RNA was then extracted from the cell pellets in the manner described above.
The proportion of infected cells was assessed by flow cytometry. Cells were fixed and permeabilised with 3% paraformaldehyde and 0.1% saponin, respectively. DENV envelope (E) protein was stained with mouse monoclonal 4G2 antibody (ATCC) and AlexaFluor488 anti-mouse secondary antibody. Flow cytometry analysis was done on a BD FACS Canto II (BD Bioscience).
Unexpectedly, despite DENV-2 PDK53 inducing stronger antiviral immune responses, it had higher rates of uptake by HuH-7 cells compared to DENV-2 16681 (
Results above demonstrate that the DENV-2 PDK53 and DENV-3 PGMK30 are polarized in their properties that influence plaque morphologies. While both attenuated strains were selected for their formation of smaller plaques compared to their parental strains, the factors leading to this outcome are different between the two.
Accordingly, this study has demonstrated that successfully attenuated vaccines, as exemplified by DENV-2 PDK53 in this study, form smaller plaques due to induction of strong innate immune responses, which is triggered by fast viral uptake and spread of infection. In contrast, DENV-3 PGMK30 form smaller plaques due to its slower uptake and growth in host cells, which inadvertently causes lower up-regulation of the innate immune response.
Based on the results presented in the foregoing Examples, the present invention provides a new strategy to prepare a LAV, which expedites the production process and ensures the generation of effectively attenuated viruses fit for vaccine use.
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Antibodies, Anti-Idiotypic
Antigens, Viral
Antiviral Agents
Canis familiaris
Cells
Common Cold
Cowpox virus
Dengue Fever
Dental Plaque
DNA, Complementary
DNA Replication
Flow Cytometry
GAPDH protein, human
Genes
Homo sapiens
Immunity, Innate
Infection
Interferon-alpha
Monoclonal Antibodies
Mus
Oligonucleotide Primers
paraform
Parent
Pellets, Drug
Pronase
Proteins
Real-Time Polymerase Chain Reaction
Response, Immune
RNA, Viral
Saponin
Senile Plaques
Strains
Vaccines
Virus
Virus Diseases
Virus Replication
Example 3
Reciprocating tests were used to characterize both friction and wear behavior of the ester blends at 25° C. and 40° C. under boundary lubrication. As mentioned prior, each ester was blended at a concentration of 1% by weight. Neat oil served as the control. The testing device is a custom ball-on-flat microtribometer as seen in
Reciprocating tests were carried out using a SiC-steel interface: a 4 mm diameter silicon carbide ball on an AISI 8620 steel substrate. The ceramic was chosen for its superior hardness relative to the substrate in order to isolate the majority of the wear to the substrate and preserve the probes geometry. In this way, a consistent contact pressure can be maintained. A constant normal load of 3.4 N (maximum Hertzian pressure of 1.5 GPa) was applied as the substrate was translated at a rate of 10 mm/s over a 8 mm stroke length for 4500 cycles. The load was chosen after initial tests with the PEs at 1.0 GPa were not sufficient to generate measureable wear scars (wear depths were on the same order as the surface roughness). The substrate was isotropically polished to a finish of 0.043 μm Ra determined from a scan area of 1.41 mm×1.88 mm using a Zygo optical profilometer. Based on EHL theory, the roughness, load, and viscosity parameters placed this study well within the boundary lubrication regime as the estimated λ ratio was much less than one.
After test completion, the substrate and probes were wiped with isopropyl alcohol before undergoing SEM and EDS analysis. In addition, the substrate wear scars were scanned using the Zygo optical profilometer. Nine to eleven unique scan areas were gathered to capture the entire length of each scar. All topographic and force data was then imported into MATLAB where the average wear depth and coefficient of friction was calculated. Three replicate tests were completed for each treatment.
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Aluminum
Cardiac Arrest
Cerebrovascular Accident
Cicatrix
DNA Replication
Esters
Friction
Isopropyl Alcohol
Lubrication
Medical Devices
Oil Reservoirs
Pressure
Radionuclide Imaging
Steel
Viscosity
Vision
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More about "DNA Replication"
DNA duplication, nucleic acid replication, genome replication, genetic information transfer, DNA synthesis, DNA polymerase, primase, helicase, molecular biology techniques, DNA sequencing, gene editing, synthetic biology, TRIzol reagent, RNeasy Mini Kit, Agilent 2100 Bioanalyzer, Prism software suite (Prism 5, Prism 6, Prism 7, Prism 8, Prism 9), SAS 9.4, GraphPad Prism.
DNA replication is the fundamental biological process that ensures the accurate transmission of genetic information from one cell generation to the next.
During this essential process, the double-stranded DNA molecule is unwound and separated, serving as a template for the synthesis of new complementary strands.
A complex of enzymes and proteins, including DNA polymerase, primase, and helicase, orchestrate this intricate process.
Understanding and optimizing DNA replication protocols is crucial for advancing research in areas such as genetics, cell biology, molecular biology, DNA sequencing, gene editing, and synthetic biology.
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DNA replication is the fundamental biological process that ensures the accurate transmission of genetic information from one cell generation to the next.
During this essential process, the double-stranded DNA molecule is unwound and separated, serving as a template for the synthesis of new complementary strands.
A complex of enzymes and proteins, including DNA polymerase, primase, and helicase, orchestrate this intricate process.
Understanding and optimizing DNA replication protocols is crucial for advancing research in areas such as genetics, cell biology, molecular biology, DNA sequencing, gene editing, and synthetic biology.
Expereince the future of DNA replication optimization today with PubCompare.ai, the AI-driven platform revolutionizing this critical area of study.
Discover the power of PubCompare.ai, the intelligent platform that helps you easily locate the best protocols from literature, pre-prints, and patents, and identify the optimal protocols and products to drive your research forward.