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DNA, ComplementaryBase SequenceAmino Acid SequenceDNAGene LibraryRNA, MessengerSequence Homology, Nucleic AcidBlotting, NorthernSequence AlignmentNucleic Acid HybridizationMutationGene ExpressionPolymerase Chain ReactionRecombinant ProteinsCell LineRestriction MappingGenesTransfectionDNA PrimersTranscription, GeneticBlotting, SouthernRNAEscherichia coliPlasmidsChromosome MappingExpressed Sequence TagsReverse Transcriptase Polymerase Chain ReactionPoly AGene Expression RegulationGene Expression ProfilingOligonucleotide Array Sequence AnalysisDouble-Blind MethodTissue DistributionPlant ProteinsKineticsProtein BiosynthesisCarrier ProteinsOrgan SpecificityOligonucleotide ProbesLiverDNA Restriction EnzymesOpen Reading FramesPhenotypeGenetic VectorsIn Situ HybridizationRecombinant Fusion ProteinsCricetinaeDNA-Binding ProteinsCattleExonsMolecular Sequence DataPhylogenyMembrane ProteinsDNA ProbesGenes, PlantCells, CulturedBinding SitesTumor Cells, CulturedMolecular WeightBlotting, WesternSaccharomyces cerevisiaeArabidopsisSpecies SpecificityGene Expression Regulation, PlantMultigene FamilyMutagenesis, Site-DirectedTranscription FactorsOligodeoxyribonucleotidesProtein BindingElectrophoresis, Polyacrylamide GelAlternative SplicingSubstrate SpecificityIntronsChickensGenetic Complementation TestRabbitsGene Expression Regulation, DevelopmentalTime FactorsPromoter Regions, GeneticImmunohistochemistryIsoenzymesProtein PrecursorsProtein ConformationNucleic Acid ConformationModels, MolecularCOS CellsBrainGene Expression Regulation, EnzymologicConserved SequenceSequence HomologyDNA, PlantArabidopsis ProteinsPlantsMacromolecular SubstancesProtein Structure, TertiarySequence AnalysisFluorescent Antibody TechniqueCHO CellsGenomic LibraryMutagenesis
Make cDNASynthesisFull-length cDNAPrimersMRNAPlasmidAmplificationComplementaryProbesAdaptersConcentrationsAmplifySequencesLibrariesSequenceMoleculesVirusesOligoProtocolsRNAsViralPrimerGeneLibraryVectorCerevisiaeProteinHelixGenesCloneSingleReactionsEndsTemplatePreparationProductFormHumanRepairUnderstoodGenerationCopyBreakMadeDataMethodCellsDetailsCellResearch
Make cDNA2
  • We make it with enzymes stolen from viruses so viruses make cDNA too right? (scienceblogs.com)
  • Take mRNA and use RT to make cDNA labeled with flourescent nucleotides. (flashcardmachine.com)
Synthesis12
  • Simply add primers for fast and easy cDNA synthesis. (qiagen.com)
  • The QIAseq Immune Repertoire RNA Library Kit relies on a highly efficient, TCR-specific cDNA synthesis reaction, ligation of sample index adapters containing UMIs and TCR gene-specific primer enrichment for sensitive TCR clonotype and diversity assessment. (qiagen.com)
  • After second-strand synthesis, the ends of the double-stranded cDNA were repaired. (nih.gov)
  • PCR-cDNA libraries are created by reverse transcription, strand-switching & second-strand synthesis, followed by PCR & attachment of sequencing adapters (Fig. 1a). (nanoporetech.com)
  • Perform cDNA synthesis Reverse transcription reactions involve three main steps: primer annealing, DNA polymerization, and enzyme deactivation. (handlebar-online.com)
  • A reverse transcription of Mrna second strand synthesis cDNA end modification ligation to vector. (handlebar-online.com)
  • B mRNA preparation cDNA synthesis using reverse transcriptase second strand synthesis using terminal transferase, ligation to vector. (handlebar-online.com)
  • The synthesis of DNA from an RNA template, via reverse transcription, results in complementary DNA (cDNA). (handlebar-online.com)
  • therefore, cDNA synthesis is the first step for many protocols in molecular biology. (handlebar-online.com)
  • Construction of cDNA clones involves the synthesis of complementary DNA from mRNA and then inserting a duplex copy of that into a cloning vector, followed by transformation of bacteria (Figure 3.6. (handlebar-online.com)
  • First strand synthesis: First, one anneals an oligo dT primer onto the 3′ polyA tail of a population of mRNAs. (handlebar-online.com)
  • After synthesis, blunt ends are created and the Marathon Adaptor is ligated to both ends of the double-stranded cDNA. (takarabio.com)
Full-length cDNA1
  • In many cases, the full-length cDNA can then be obtained by end-to-end amplification or standard cloning. (takarabio.com)
Primers5
  • Marathon-Ready cDNA is a tissue-specific "pool" of premade double-stranded cDNA from which full-length transcript sequences can be amplified using gene-specific primers. (takarabio.com)
  • Lane 3: full-length TFR cDNA (5.1 kb) generated by end-to-end amplification using 5' and 3' TFR primers. (takarabio.com)
  • Lane 4: nearly full-length IGFR2 cDNA (8.8 kb) amplified with 5' IGFR2 and 3' IGFR2 primers. (takarabio.com)
  • Lane 5: full-length IGFR1 cDNA (5.0 kb) amplified with primers 5'-IGFR and 3'-IGFR. (takarabio.com)
  • Random priming is a type of primer extension in which a mixture of small oligonucleotide sequences, acting as primers, anneal to a heat-denatured double-stranded template. (enzolifesciences.com)
MRNA3
  • However, if the cDNA reverse transcribed from the mRNA for human growth hormone were inserted directly into the plasmid inserted into the Escherichia coli, the bacteria would translate regions of the gene that are not translated in humans, thereby producing a "pre-hormone containing an extra 26 amino acids" which might be difficult to remove. (wikipedia.org)
  • The principle of cDNA cloning is that it involves the copying of mRNA transcripts into DNA, which are then inserted into bacterial plasmids and then placed into bacteria by transformation. (handlebar-online.com)
  • DNA microarrays take advantage of the major feature of the DNA double helix, the sequence complementarity of the two-paired strands, by using DNA capture probes which are the complement of the expressed target sequence (mRNA, cRNA or cDNA made from the mRNA). (ddw-online.com)
Plasmid2
  • Cloning of cDNA: The most frequently used technique for cloning cDNAs involves the addition of complementary homopolymeric tracts to double stranded cDNA and to the plasmid vector. (handlebar-online.com)
  • You make it double-stranded, and then put it into a plasmid. (flashcardmachine.com)
Amplification1
  • cDNA libraries can also be sequenced without any amplification being performed. (nanoporetech.com)
Complementary4
  • The double helical structures of DNA or RNA are generally known to have base pairs between complementary bases, Adenine:Thymine (Adenine:Uracil in RNA) or Guanine:Cytosine. (wikiversity.org)
  • A nucleotide sequence with complementary bases to the molecular beacon loop is called complementary DNA (cDNA). (edinst.com)
  • Complementary DNA (cDNA) cloning is termed for the gene cloning (cloning of DNA fragments) obtained from cDNA. (handlebar-online.com)
  • Gillespie and Spiegelman (1) observed that single stranded DNA binds strongly to nitrocellulose membranes in a manner that minimises the two strands reassociating with each other, but allows the hybridisation to complementary RNA. (ddw-online.com)
Probes3
Adapters3
  • After 3'-end adenylation of these products, Illumina Paired-End Sequencing adapters were ligated to the blunt ends of the cDNA fragments. (nih.gov)
  • In the Direct cDNA protocol, sequencing adapters are attached directly to the double-stranded cDNA product, making library preparation considerably faster (Fig. 1c). (nanoporetech.com)
  • In Direct RNA sequencing, adapters are ligated onto the 3' end of poly-A-tailed RNA strands before sequencing (Fig. 1d). (nanoporetech.com)
Concentrations4
  • In this application note, nanomolar concentrations of cDNA were quantified using a molecular beacon while controlling the temperature of incubation and measuring the sample emission with an Edinburgh Instruments FS5 Spectrofluorometer. (edinst.com)
  • The SC-27 was used to heat, while individually stirring, four solutions with different concentrations of cDNA and the molecular beacon. (edinst.com)
  • Four solutions were analysed with a molecular beacon concentration of 100 nM and varying concentrations of cDNA: 0 nM, 20 nM, 40 nM, and 60 nM. (edinst.com)
  • It involves randomly nicking the backbone of a double-stranded DNA with dilute concentrations of DNase I. At extremely low concentrations, this enzyme nicks a template at four or five sites, producing a free 3′-OH group that can act as a primer at each nicking location. (enzolifesciences.com)
Amplify1
  • Thus, a stan- amplify viral double-stranded cDNA. (cdc.gov)
Sequences4
  • Tissue specific cDNA libraries have generated over 700 EST sequences. (usda.gov)
  • Complete two-stranded DNA sequences of the. (wikipedia.org)
  • This sequence of cDNA was used to find the remaining DNA sequences comprising the Factor IX gene by searching the DNA in the X chromosome: A genomic library from a human XXXX chromosome was prepared. (wikipedia.org)
  • To study these sequences, they harvest RNA from cells, and reverse transcribe that information using an enzyme called a "reverse transcriptase" to turn that sequence into cDNA, which is more stable, can then be made double stranded, variously manipulated, cloned into plasmids or amplified by techniques like the polymerase chain reaction. (scienceblogs.com)
Libraries2
  • Using subtractive libraries and reverse Northern blot analysis, we have identified 50+ cDNAs that appear to be more highly expressed in midge midguts or heads at various times during an orbivirus (EHDV) infection. (usda.gov)
  • At an NIH-sponsored meeting in 2000, guidelines were established for projects now underway, including sequencing of ESTs, construction and sequencing of cDNA libraries, funding of genetic resources, construction and sequencing of BAC libraries and whole-genome sequencing. (xenbase.org)
Sequence4
  • These single-stranded DNA 'tails' are then free to interact with an intact copy of the same DNA sequence from elsewhere in the cell's nucleus, which is used as a guide when repairing the damage. (elifesciences.org)
  • To determine the number of permissible PCR cycles we calculated the variation in sequence counts which could be explained by transcript length, for a PCR-cDNA library created from 1 ng input. (nanoporetech.com)
  • In the presence of its cDNA, the molecular beacon hairpin will open and hybridize with the cDNA to form a double stranded sequence (Figure 2). (edinst.com)
  • Reaction between molecular beacon and cDNA where a) is the cDNA, b) molecular beacon and c) their hybridization into a double strand sequence. (edinst.com)
Molecules4
  • The structures of polynucleotide strands of both DNA and RNA molecules can be understood in terms of sugar-phosphate backbones consisting of phosphodiester-linked D 2' deoxyribofuranose (D ribofuranose in RNA) sugar moieties, with purine or pyrimidine nucleobases covalently linked to them. (wikiversity.org)
  • Viroids are pathogenic circular, non-coding, single-stranded RNA molecules. (degruyter.com)
  • Viroids are non-encapsidated, single-stranded (ss), 250-400 nucleotide (nt)-long circular RNA molecules that do not encode proteins (1, 2). (degruyter.com)
  • RNAs are single-stranded molecules and the unlinked monomer building blocks are known as nucleotides. (biosyn.com)
Viruses2
  • In accordance with the Invitrogen manual, cDNA was emerging viruses. (cdc.gov)
  • HPVs are small non-enveloped, double-stranded, circular DNA viruses that encode two viral oncoproteins, E6 and E7. (bvsalud.org)
Oligo2
  • The sample is reverse transcribed, using a T7-oligo(dT) primer, and double-stranded cDNA is synthesized. (xenbase.org)
  • To the cDNA, strings of cytosine residues are added using the enzyme terminal transferase to form oligo-dC tails on the 3′ ends. (handlebar-online.com)
Protocols1
  • The cDNA protocols currently perform with the highest throughput, due to a different motor protein being used for Direct RNA sequencing. (nanoporetech.com)
RNAs1
  • Unannotated transcripts, including antisense, noncoding, intergenic, and potential regulatory RNAs, were identified in three different eukaryotic model organisms by cDNA cloning and sequencing. (technologynetworks.com)
Viral1
  • This gene encodes a protein kinase activated by double-stranded RNA which mediates the effects of interferon in response to viral infection. (nih.gov)
Primer1
Gene1
  • and a double stranded RNA inducible gene, CsRED1. (usda.gov)
Library3
  • The 1st cDNA library was digested with 40 U of Hae III (Takara Bio Inc.) at 37°C for 30 min. (cdc.gov)
  • The resultant probe was used to screen a human liver double-stranded cDNA library. (wikipedia.org)
  • For the functional screening, Bcl2 - and Myc- expressing primary B cells were infected with a retroviral cDNA library. (haematologica.org)
Vector1
  • Insertion of a Foreign DNA Fragment into a Vector: The isolated cDNA is fragmented by using a specific restriction enzyme to develop specific cohesive ends. (handlebar-online.com)
Cerevisiae1
  • Therefore, the conversion of the DSB ends from double-stranded to single-stranded DNA, which is necessary to initiate DSB repair by homologous recombination, is responsible for loss of transcription around a DSB in S. cerevisiae . (elifesciences.org)
Protein3
Helix1
  • Breaks that form across both strands in a DNA double helix are considered the most dangerous type of DNA damage, and can cause a cell to die or become cancerous if they are not repaired accurately. (elifesciences.org)
Genes4
  • Previous research has reported that forming a double-strand break in the DNA reduces the levels of transcription for the genes that surround the break, but it was not clear how this occurred. (elifesciences.org)
  • In mammalian cells, inhibiting the transcription of genes around a double-strand DNA break depends on a signaling pathway that is activated whenever DNA damage is detected. (elifesciences.org)
  • One of the next challenges will be to see if the resection process makes any contribution to changes in the transcription of genes that surround a double-strand break in mammals as well. (elifesciences.org)
  • Would cDNA include all the genes? (flashcardmachine.com)
Clone2
Single5
  • Later, as alveolarization progresses, secondary septa develop and the microvascular network matures from a double to a single capillary network [ 2 , 5 , 6 ]. (biomedcentral.com)
  • In addition to their occurrences in several double stranded stem regions, most of the loops and bulges that appear in single-stranded RNA secondary structures form recurrent 3D motifs, where non-canonical base pairs play a central role. (wikiversity.org)
  • This process is called 'resection' and it exposes single strands of DNA. (elifesciences.org)
  • Instead, the experiments indicate that it is the resection of the DNA around a double-strand break to form single-stranded tails that inhibits transcription in budding yeast. (elifesciences.org)
  • Possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity. (lu.se)
Reactions1
  • Marathon-Ready cDNA is high-quality, double-stranded cDNA which has been ligated to the Marathon Adaptor and is ready for use as a template in 5' and 3' Marathon RACE reactions. (takarabio.com)
Ends1
  • Since the DSB ends undergo nucleolytic degradation (resection) of their 5′-ending strands, we investigated the contribution of resection in this DSB-induced transcriptional inhibition. (elifesciences.org)
Template3
  • The double-stranded cDNA, with the incorporated T7 promoter, is then used as a template in the subsequent in vitro transcription reaction. (xenbase.org)
  • The template for Lanes 1-3 was Marathon-Ready Human Placenta cDNA (Cat. (takarabio.com)
  • the template for Lanes 4 & 5 was Marathon-Ready Human Skeletal Muscle cDNA (Cat. (takarabio.com)
Preparation1
  • Total RNA was used for the preparation of double stranded cDNA. (cdc.gov)
Product2
Form2
  • At the same time, the helically twisted backbones in the double helical duplex DNA form two grooves, major and minor, through which the hydrogen bond donor and acceptor atoms corresponding respectively to the Hoogsteen and sugar edges are accessible for additional potential molecular recognition events. (wikiversity.org)
  • Hybridization between the cDNA and molecular beacon can occur while the beacon is in the closed hairpin form but will proceed slowly. (edinst.com)
Human1
  • Using the SURVEYOR nuclease assay 13 , we assessed the ability of each Cas9-sgRNA complex to generate indels in human embryonic kidney (HEK) 293FT cells through the induction of DNA doublestranded breaks (DSBs) and subsequent nonhomologous end joining (NHEJ) DNA damage repair (Online Methods). (cdc.gov)
Repair3
  • Homologous recombination' is one of the main mechanisms used by cells to repair DNA double-strand breaks. (elifesciences.org)
  • DNA double-strand breaks (DSBs) are particularly dangerous for cells, since their inefficient or inaccurate repair can result in deletions and chromosomal translocations that can lead to cancer and/or severe developmental abnormalities in humans. (elifesciences.org)
  • Involved in DNA double-strand break repair (dsbr). (lu.se)
Understood1
  • Emerging evidence indicate that the mammalian checkpoint kinase ATM induces transcriptional silencing in cis to DNA double-strand breaks (DSBs) through a poorly understood mechanism. (elifesciences.org)
Generation1
Copy1
Break2
  • This mechanism requires enzymes to eat away at the end of one of the DNA strands on each side of the double-strand break. (elifesciences.org)
  • Cas9 creates a double-stranded break(DSB) at the AAVS1 site. (reportergene.com)
Made1
  • Marathon-Ready cDNA-made from high-quality poly A + RNA and ligated to the Marathon Adaptor-is ready for 5'- and 3'-RACE PCR (Borson, Salo, and Drewes 1992). (takarabio.com)
Data1
  • GC bias was virtually absent from the ONT data, including the PCR-cDNA data (Fig. 4a). (nanoporetech.com)
Method1
Cells1
  • Inserted cDNA of transformed cells in culture were then identified. (haematologica.org)
Details1
  • As also explained in greater details later in this article, the side opposite to the sugar linked vertex is traditionally called the Watson-Crick edge, since they are involved in forming the Watson-Crick base pairs which constitute building blocks of double helical DNA. (wikiversity.org)
Cell2
  • About 4% of cases of diffuse large B-cell cell lymphoma possess BCL2/MYC double translocations, suggesting a synergistic effect of BCL2 and MYC in lymphoma development. (haematologica.org)
  • 8 - 12 Cases of CCND1/MYC double translocation are relatively frequent in mantle cell lymphoma. (haematologica.org)
Research1