DNA Primers: Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques.Templates, Genetic: Macromolecular molds for the synthesis of complementary macromolecules, as in DNA REPLICATION; GENETIC TRANSCRIPTION of DNA to RNA, and GENETIC TRANSLATION of RNA into POLYPEPTIDES.Base Sequence: The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.HIV Reverse Transcriptase: A reverse transcriptase encoded by the POL GENE of HIV. It is a heterodimer of 66 kDa and 51 kDa subunits that are derived from a common precursor protein. The heterodimer also includes an RNAse H activity (RIBONUCLEASE H, HUMAN IMMUNODEFICIENCY VIRUS) that plays an essential role the viral replication process.RNA-Directed DNA Polymerase: An enzyme that synthesizes DNA on an RNA template. It is encoded by the pol gene of retroviruses and by certain retrovirus-like elements. EC 2.7.7.49.Ribonuclease H: A ribonuclease that specifically cleaves the RNA moiety of RNA:DNA hybrids. It has been isolated from a wide variety of prokaryotic and eukaryotic organisms as well as RETROVIRUSES.Euplotes: A genus of ciliate protozoa having a dorsoventrally flattened body with widely spaced rows of short bristle-like cilia on the dorsal surface.Deoxyguanine Nucleotides: Guanine nucleotides which contain deoxyribose as the sugar moiety.Molecular Sequence Data: Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.Polymerase Chain Reaction: In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.DNA-Directed DNA Polymerase: DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair.RNA: A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed)DNA: A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).Dideoxynucleotides: The phosphate esters of DIDEOXYNUCLEOSIDES.DNA Replication: The process by which a DNA molecule is duplicated.Telomerase: An essential ribonucleoprotein reverse transcriptase that adds telomeric DNA to the ends of eukaryotic CHROMOSOMES.Aphidicolin: An antiviral antibiotic produced by Cephalosporium aphidicola and other fungi. It inhibits the growth of eukaryotic cells and certain animal viruses by selectively inhibiting the cellular replication of DNA polymerase II or the viral-induced DNA polymerases. The drug may be useful for controlling excessive cell proliferation in patients with cancer, psoriasis or other dermatitis with little or no adverse effect upon non-multiplying cells.DNA, Single-Stranded: A single chain of deoxyribonucleotides that occurs in some bacteria and viruses. It usually exists as a covalently closed circle.DNA, Viral: Deoxyribonucleic acid that makes up the genetic material of viruses.DNA Polymerase I: A DNA-dependent DNA polymerase characterized in prokaryotes and may be present in higher organisms. It has both 3'-5' and 5'-3' exonuclease activity, but cannot use native double-stranded DNA as template-primer. It is not inhibited by sulfhydryl reagents and is active in both DNA synthesis and repair. EC 2.7.7.7.Nucleic Acid Conformation: The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.Nucleotides: The monomeric units from which DNA or RNA polymers are constructed. They consist of a purine or pyrimidine base, a pentose sugar, and a phosphate group. (From King & Stansfield, A Dictionary of Genetics, 4th ed)Substrate Specificity: A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.Reverse Transcriptase Inhibitors: Inhibitors of reverse transcriptase (RNA-DIRECTED DNA POLYMERASE), an enzyme that synthesizes DNA on an RNA template.Kinetics: The rate dynamics in chemical or physical systems.RNA, Viral: Ribonucleic acid that makes up the genetic material of viruses.Mutation: Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.Binding Sites: The parts of a macromolecule that directly participate in its specific combination with another molecule.Escherichia coli: A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.HIV-1: The type species of LENTIVIRUS and the etiologic agent of AIDS. It is characterized by its cytopathic effect and affinity for the T4-lymphocyte.Sequence Analysis, DNA: A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis.DNA, Bacterial: Deoxyribonucleic acid that makes up the genetic material of bacteria.Random Amplified Polymorphic DNA Technique: Technique that utilizes low-stringency polymerase chain reaction (PCR) amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments. RAPD technique may be used to determine taxonomic identity, assess kinship relationships, analyze mixed genome samples, and create specific probes.Sensitivity and Specificity: Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed)Cloning, Molecular: The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.RNA, Transfer, Lys: A transfer RNA which is specific for carrying lysine to sites on the ribosomes in preparation for protein synthesis.Phylogeny: The relationships of groups of organisms as reflected by their genetic makeup.Amino Acid Sequence: The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.RNA, Ribosomal, 16S: Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis.Species Specificity: The restriction of a characteristic behavior, anatomical structure or physical system, such as immune response; metabolic response, or gene or gene variant to the members of one species. It refers to that property which differentiates one species from another but it is also used for phylogenetic levels higher or lower than the species.DNA, Ribosomal: DNA sequences encoding RIBOSOMAL RNA and the segments of DNA separating the individual ribosomal RNA genes, referred to as RIBOSOMAL SPACER DNA.Transcription, Genetic: The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.DNA Primase: A single-stranded DNA-dependent RNA polymerase that functions to initiate, or prime, DNA synthesis by synthesizing oligoribonucleotide primers. EC 2.7.7.-.Methacrylates: Acrylic acids or acrylates which are substituted in the C-2 position with a methyl group.Sequence Homology, Nucleic Acid: The sequential correspondence of nucleotides in one nucleic acid molecule with those of another nucleic acid molecule. Sequence homology is an indication of the genetic relatedness of different organisms and gene function.Dental Bonding: An adhesion procedure for orthodontic attachments, such as plastic DENTAL CROWNS. This process usually includes the application of an adhesive material (DENTAL CEMENTS) and letting it harden in-place by light or chemical curing.Genes, Bacterial: The functional hereditary units of BACTERIA.Nucleic Acid Amplification Techniques: Laboratory techniques that involve the in-vitro synthesis of many copies of DNA or RNA from one original template.Resin Cements: Dental cements composed either of polymethyl methacrylate or dimethacrylate, produced by mixing an acrylic monomer liquid with acrylic polymers and mineral fillers. The cement is insoluble in water and is thus resistant to fluids in the mouth, but is also irritating to the dental pulp. It is used chiefly as a luting agent for fabricated and temporary restorations. (Jablonski's Dictionary of Dentistry, 1992, p159)Sequence Alignment: The arrangement of two or more amino acid or base sequences from an organism or organisms in such a way as to align areas of the sequences sharing common properties. The degree of relatedness or homology between the sequences is predicted computationally or statistically based on weights assigned to the elements aligned between the sequences. This in turn can serve as a potential indicator of the genetic relatedness between the organisms.Oligonucleotides: Polymers made up of a few (2-20) nucleotides. In molecular genetics, they refer to a short sequence synthesized to match a region where a mutation is known to occur, and then used as a probe (OLIGONUCLEOTIDE PROBES). (Dorland, 28th ed)Oligonucleotide Probes: Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin.Taq Polymerase: A heat stable DNA-DIRECTED DNA POLYMERASE from the bacteria Thermus aquaticus. It is widely used for the amplification of genes through the process of POLYMERASE CHAIN REACTION. EC 2.7.7.-.DNA Fingerprinting: A technique for identifying individuals of a species that is based on the uniqueness of their DNA sequence. Uniqueness is determined by identifying which combination of allelic variations occur in the individual at a statistically relevant number of different loci. In forensic studies, RESTRICTION FRAGMENT LENGTH POLYMORPHISM of multiple, highly polymorphic VNTR LOCI or MICROSATELLITE REPEAT loci are analyzed. The number of loci used for the profile depends on the ALLELE FREQUENCY in the population.RNA, Messenger: RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.DNA Probes: Species- or subspecies-specific DNA (including COMPLEMENTARY DNA; conserved genes, whole chromosomes, or whole genomes) used in hybridization studies in order to identify microorganisms, to measure DNA-DNA homologies, to group subspecies, etc. The DNA probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the DNA probe include the radioisotope labels 32P and 125I and the chemical label biotin. The use of DNA probes provides a specific, sensitive, rapid, and inexpensive replacement for cell culture techniques for diagnosing infections.Electrophoresis, Agar Gel: Electrophoresis in which agar or agarose gel is used as the diffusion medium.DNA, Plant: Deoxyribonucleic acid that makes up the genetic material of plants.Microsatellite Repeats: A variety of simple repeat sequences that are distributed throughout the GENOME. They are characterized by a short repeat unit of 2-8 basepairs that is repeated up to 100 times. They are also known as short tandem repeats (STRs).Genotype: The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS.RNA, Bacterial: Ribonucleic acid in bacteria having regulatory and catalytic roles as well as involvement in protein synthesis.DNA, Complementary: Single-stranded complementary DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase. cDNA (i.e., complementary DNA, not circular DNA, not C-DNA) is used in a variety of molecular cloning experiments as well as serving as a specific hybridization probe.Polymorphism, Restriction Fragment Length: Variation occurring within a species in the presence or length of DNA fragment generated by a specific endonuclease at a specific site in the genome. Such variations are generated by mutations that create or abolish recognition sites for these enzymes or change the length of the fragment.Nucleic Acid Hybridization: Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503)DNA, Fungal: Deoxyribonucleic acid that makes up the genetic material of fungi.Genetic Variation: Genotypic differences observed among individuals in a population.Shear Strength: The internal resistance of a material to moving some parts of it parallel to a fixed plane, in contrast to stretching (TENSILE STRENGTH) or compression (COMPRESSIVE STRENGTH). Ionic crystals are brittle because, when subjected to shear, ions of the same charge are brought next to each other, which causes repulsion.Reverse Transcriptase Polymerase Chain Reaction: A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols.Blotting, Southern: A method (first developed by E.M. Southern) for detection of DNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES.Genetic Markers: A phenotypically recognizable genetic trait which can be used to identify a genetic locus, a linkage group, or a recombination event.Bacterial Proteins: Proteins found in any species of bacterium.Amphetamine: A powerful central nervous system stimulant and sympathomimetic. Amphetamine has multiple mechanisms of action including blocking uptake of adrenergics and dopamine, stimulation of release of monamines, and inhibiting monoamine oxidase. Amphetamine is also a drug of abuse and a psychotomimetic. The l- and the d,l-forms are included here. The l-form has less central nervous system activity but stronger cardiovascular effects. The d-form is DEXTROAMPHETAMINE.Sequence Homology, Amino Acid: The degree of similarity between sequences of amino acids. This information is useful for the analyzing genetic relatedness of proteins and species.Polymorphism, Genetic: The regular and simultaneous occurrence in a single interbreeding population of two or more discontinuous genotypes. The concept includes differences in genotypes ranging in size from a single nucleotide site (POLYMORPHISM, SINGLE NUCLEOTIDE) to large nucleotide sequences visible at a chromosomal level.DNA, Ribosomal Spacer: The intergenic DNA segments that are between the ribosomal RNA genes (internal transcribed spacers) and between the tandemly repeated units of rDNA (external transcribed spacers and nontranscribed spacers).Ankle Brachial Index: Comparison of the BLOOD PRESSURE between the BRACHIAL ARTERY and the POSTERIOR TIBIAL ARTERY. It is a predictor of PERIPHERAL ARTERIAL DISEASE.Dentin-Bonding Agents: Cements that act through infiltration and polymerization within the dentinal matrix and are used for dental restoration. They can be adhesive resins themselves, adhesion-promoting monomers, or polymerization initiators that act in concert with other agents to form a dentin-bonding system.RNA Nucleotidyltransferases: Enzymes that catalyze the template-directed incorporation of ribonucleotides into an RNA chain. EC 2.7.7.-.Gene Library: A large collection of DNA fragments cloned (CLONING, MOLECULAR) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences.Oligodeoxyribonucleotides: A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.

Activation of systemic acquired silencing by localised introduction of DNA. (1/43244)

BACKGROUND: In plants, post-transcriptional gene silencing results in RNA degradation after transcription. Among tobacco transformants carrying a nitrate reductase (Nia) construct under the control of the cauliflower mosaic virus 35S promoter (35S-Nia2), one class of transformants spontaneously triggers Nia post-transcriptional gene silencing (class II) whereas another class does not (class I). Non-silenced plants of both classes become silenced when grafted onto silenced stocks, indicating the existence of a systemic silencing signal. Graft-transmitted silencing is maintained in class II but not in class I plants when removed from silenced stocks, indicating similar requirements for spontaneous triggering and maintenance. RESULTS: Introduction of 35S-Nia2 DNA by the gene transfer method called biolistics led to localised acquired silencing (LAS) in bombarded leaves of wild-type, class I and class II plants, and to systemic acquired silencing (SAS) in class II plants. SAS occurred even if the targeted leaf was removed 2 days after bombardment, indicating that the systemic signal is produced, transmitted and amplified rapidly. SAS was activated by sense, antisense and promoterless Nia2 DNA constructs, indicating that transcription is not required although it does stimulate SAS. CONCLUSIONS: SAS was activated by biolistic introduction of promoterless constructs, indicating that the DNA itself is a potent activator of post-transcriptional gene silencing. The systemic silencing signal invaded the whole plant by cell-to-cell and long-distance propagation, and reamplification of the signal.  (+info)

Impaired translesion synthesis in xeroderma pigmentosum variant extracts. (2/43244)

Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3' end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage process.  (+info)

Overexpression of CuZn superoxide dismutase protects RAW 264.7 macrophages against nitric oxide cytotoxicity. (3/43244)

Initiation of nitric oxide (NO.)-mediated apoptotic cell death in RAW 264.7 macrophages is associated with up-regulation of mitochondrial manganese superoxide dismutase (MnSOD; SOD2) and down-regulation of cytosolic copper zinc superoxide dismutase (CuZnSOD; SOD1) at their individual mRNA and protein levels. To evaluate the decreased CuZnSOD expression and the initiation of apoptosis we stably transfected macrophages to overexpress human CuZnSOD. Individual clones revealed a 2-fold increase in CuZnSOD activity. Expression of a functional and thus protective CuZnSOD was verified by attenuated superoxide (O2(.)-)-mediated apoptotic as well as necrotic cell death. In this study we showed that SOD-overexpressing macrophages (R-SOD1-12) were also protected against NO.-initiated programmed cell death. Protection was substantial towards NO. derived from exogenously added NO donors or when NO. was generated by inducible NO synthase activation, and was evident at the level of p53 accumulation, caspase activation and DNA fragmentation. Stimulation of parent and SOD-overexpressing cells with a combination of lipopolysaccharide and murine interferon gamma produced equivalent amounts of nitrite/nitrate, which ruled out attenuated inducible NO. synthase activity during protection. Because protection by a O2(.)--scavenging system during NO. -intoxication implies a role of NO. and O2(.)- in the progression of cell damage, we used uric acid to delineate the role of peroxynitrite during NO.-elicited apoptosis. The peroxynitrite scavenger uric acid left S-nitrosoglutathione or spermine-NO-elicited apoptosis unaltered, blocking only 3-morpholinosydnonimine-mediated cell death. As a result we exclude peroxynitrite from contributing, to any major extent, to NO. -mediated apoptosis. Therefore protection observed with CuZnSOD overexpression is unlikely to stem from interference with peroxynitrite formation and/or action. Unequivocally, the down-regulation of CuZnSOD is associated with NO. cytotoxicity, whereas CuZnSOD overexpression protects macrophages from apoptosis.  (+info)

Co-expression of glutathione S-transferase with methionine aminopeptidase: a system of producing enriched N-terminal processed proteins in Escherichia coli. (4/43244)

We describe here an Escherichia coli expression system that produces recombinant proteins enriched in the N-terminal processed form, by using glutathione S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to chick and rat respectively. Approximately 90% of the cGSTM1-1 or rGSTT1-1 overexpressed in E. coli under the control of a phoA promoter retained the initiator methionine residue that was absent from the mature isoenzymes isolated from tissues. The amount of initiator methionine was decreased to 40% of the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogenous methionine aminopeptidase gene under the control of a separate phoA promoter. The recombinant proteins expressed were mainly methionine aminopeptidase. The yield of cGSTM1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene. By replacing the phoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically. The yield of the co-expressed cGSTM1-1 was approx. 60% of that in the absence of the exogenous methionine aminopeptidase gene; approx. 65% of the initiator methionine residues were removed from the enzyme. Under similar conditions, N-terminal processing was observed in approx. 70% of the recombinant rGSTT1-1 expressed. By increasing the concentration of phosphate in the growth medium, the amount of initiator methionine on cGSTM1-1 was decreased to 14% of the overexpressed isoenzymes, whereas no further improvement could be observed for rGSTT1-1. The initiator methionine residue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1-1. However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugating activity of the purified recombinant rGSTT1-1 are markedly higher that those reported recently for the same isoenzyme isolated from rat livers.  (+info)

Differential regulation of the human nidogen gene promoter region by a novel cell-type-specific silencer element. (5/43244)

Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms.  (+info)

Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells. (6/43244)

To investigate which retinoid receptors are critical in the regulation by all-trans-retinoic acid (RA) of the mucin genes MUC2, MUC5AC and MUC5B in cultured normal human tracheobronchial epithelial (NHTBE) cells, we used pan-RAR-, pan-RXR- and RAR- isotype (alpha, beta and gamma)-selective agonists and RARalpha- and RARgamma-selective antagonists (RAR is RA receptor and RXR is retinoid X receptor). RAR-, RARalpha- and RARgamma-selective agonists strongly induced mucin mRNAs in a dose-dependent manner, while the RARbeta-selective retinoid only weakly induced mucin gene expression at very high concentrations (1 microM). The pan-RXR-selective agonist by itself did not induce mucin gene expression, but acted synergistically with suboptimal concentrations of the pan-RAR agonist. A retinoid with selective anti-activator-protein-1 activity only marginally induced mucin gene expression. The RARalpha antagonist strongly inhibited mucin gene induction and mucous cell differentiation caused by RA and by the RARalpha- and RARgamma-selective retinoids. In contrast, the RARgamma antagonist only weakly inhibited RARalpha-selective-retinoid-induced mucin gene expression, but completely blocked mucin gene expression induced by the RARgamma-selective retinoid. Our studies indicate that RARalpha is the major retinoid receptor subtype mediating RA-dependent mucin gene expression and mucous cell differentiation, but that the RARgamma isotype can also induce mucin genes. Furthermore these studies suggest that RARbeta is probably not (directly) involved in RA-induced mucin gene expression.  (+info)

Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells. (7/43244)

Under conditions associated with local and systemic inflammation, mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of nitric oxide (NO) in the glomerulus. To further define the mechanisms of NO action in the glomerulus, we attempted to identify genes which are regulated by NO in rat glomerular mesangial cells. We identified vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase (FLT-1) to be under the regulatory control of exogenously applied NO in these cells. Using S-nitroso-glutathione (GSNO) as an NO-donating agent, VEGF expression was strongly induced, whereas expression of its FLT-1 receptor simultaneously decreased. Expressional regulation of VEGF and FLT-1 mRNA was transient and occurred rapidly within 1-3 h after GSNO treatment. Expression of a second VEGF-specific receptor, fetal liver kinase-1 (FLK-1/KDR), could not be detected. The inflammatory cytokine interleukin-1beta mediated a moderate increase in VEGF expression after 24 h and had no influence on FLT-1 expression. In contrast, platelet-derived growth factor-BB and basic fibroblast growth factor had no effect on VEGF expression, but strongly induced FLT-1 mRNA levels. Obviously, there is a differential regulation of VEGF and its receptor FLT-1 by NO, cytokines and growth factors in rat mesangial cells.  (+info)

Comparison of the fibrin-binding activities in the N- and C-termini of fibronectin. (8/43244)

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction.  (+info)

This month in Alkami PCR Reviews, Lance Larka talks about free online primer design software. Featured in June are DOPE2, DoPrimer, NetPrimer, and Oligos-U-Like/Primers3. His article is at: http://www.alkami.com/reviews/rvwdsgn3.htm Our current list of primer tool links and brief descriptions that we have at http://www.alkami.com/primers/refdsgn.htm includes: * CODEHOP * DOPE2 * DoPrimer * The GPRIME Package * NetPrimer * Oligos-U-Like * Primer Design * Primer3 * Primers! * Primers! Lite * Primer Selection * STS Pipeline v1.2 * The Primer Generator * Web Primer (Stanford) * Web Primers * WWW GeneFisher * xprimer Other primer design resources are listed at http://www.alkami.com/primers/idxprmr.htm The Alkami Biosystems site, http://www.alkami.com, is a comprehensive resource for the polymerase chain reaction. We have online references that include primer design tips and tools, reagent concentrations, polymerase characteristics, PCR methods, and temperature calculations. In addition, you will find ...
Primer Design for PCR: Primer guidelines page offers a look at the general and useful guidelines laid for designing primers for PCR reaction including Primer Tm considerations, PCR primer cross dimer values, annealing temperature and primer GC%
In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3 end of primed double stranded DNA, the binding of the 3 end of the primer is especially important, while the 5 end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3 to the 5 end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3 base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3 to 5 order to fabricate the ...
In general, primers are designed to identify specific locations within a long region of DNA, either plasmid or genomic. Primer binding sites are ideally unique within the range of DNA found in the reaction tube. Single primers are used to amplify and label DNA fragments for sequencing reactions, or as probes for Southern blots. Pairs of primers are used to delimit the range of DNA amplified during a PCR reaction. Because biological enzymes selectively add bases on the 3 end of primed double stranded DNA, the binding of the 3 end of the primer is especially important, while the 5 end of the primer can either bind or not, with relatively little effect on most uses of the primer. The binding of the 4-e 3 to the 5 end, the opposite from the DNA synthesis direction found in biological systems. Synthesis starts with a glass bead filled column containing a specific 3 base bound to the glass. Chemically activated nucleotides, termed phosphoramidites, are linked in 3 to 5 order to fabricate the ...
Download Primer3 - PCR primer design tool for free. Design PCR primers from DNA sequence. Widely used (190k Google hits for primer3).
Dear All, I searched for an 18S rRNA gene of an organism on the NCBI website. I found the sequence and designed primers using the primer tool on the same website. Now, I want to check the primer specificity (i.e. can I use this primer as an identification tool to detect a specific species using PCR) and the question is: do I need to BLAST the primer sequence (both forward and reverse) or to BLAST the sequence of the amplified fragment (PCR product). Many thanks in advance. Best regards Mohammad Email: moh_aldeeb from yahoo.com Personal Website: http://sites.google.com/site/draldeebsite/ --- On Wed, 7/6/11, mnr mnr ,mnr475 from gmail.com, wrote: , From: mnr mnr ,mnr475 from gmail.com, , Subject: Blunt end cloning , To: methods from magpie.bio.indiana.edu , Date: Wednesday, July 6, 2011, 1:23 PM , Hi all. , , I have tried cloning a potential toxic gene about 1.2 kbp , into E.coli with , T7 promoter. After two months, I am still not successful. I , am now plannig , to do cloning into pUC18 vector ...
(KudoZ) English to Czech translation of Oligos/Primers/Probes: Oligonukleotidy / primery / sondy [Biochemie - Chemistry; Chem Sci/Eng (Science)].
Sequencing Primers and Plasmids - posted in Molecular Biology: Im a little confused about the notation of the sequencing primers listed on the plasmid map for Invitrogens pCR 2.1 and 4.0 used in TOPO TA cloning. Why is the forward primer 3 insertion site while the reverse primer 5 insertion. I thought it is opposite of that. Looking for some explanation. Wondering if Im looking at the map wrong, or if Im not understanding something. Link for map is below.http:...
GENEWIZ offers a variety of free universal primers for sequencing. These free universal primers are being updated to reflect the needs of our customers. Users in our new CLIMS Online Ordering and Data Management System have access to the Updated GENEWIZ Universal Primer list (see below). If you are unsure which CLIMS system you are using, please contact our Technical Support team at 877-GENEWIZ, ext. 2. ...
OLIGO performs a range of functions for researchers in PCR and related technologies, enables to design consensus, multiplex and degenerate primers. Reverse translation, restriction enzyme and open reading frames analysis, oligonucleotide database, primer secondary structure, LCR, siRNA, molecular beacons and nested primers design, real time PCR, batch file processing.
The reasons for primer dimer formation in an NTC are often due to multiple factors. These include: sub-optimal primer annealing temperature (often due to differences in buffering conditions between different qPCR kits), sub-optimal primer synthesis (HPLC-purified primers result in less primer dimer formation and are useful for low copy number detection), and poor primer design (using software such as Primer3 is recommended when designing primers). An alternative is to reduce the total number of cycles in a qPCR reaction if amplification of the primer dimer lies outside of the range of the experimental data eg. if the sample being tested has an average Cq of 28 cycles and contains specific product only (no primer dimer), and the NTC amplifies a primer dimer after 37 cycles, the reaction can be run for 35 cycles, rather than 40. This approach can be taken only when the sample being tested gives rise to a specific product and not a combination of specific product and primer dimer, and also when the ...
RT-PCR primer design - posted in PCR, RT-PCR and Real-Time PCR: Hi, I want to ask about how to design primer for RT-PCR. Specifically, i want to design primer based on the human mRNA encoding the c-myc, p53, caspase-3 and bcl-2 gene. I have no clue where to start and what to do... thank you..
( http://www.abnova.com ) - Primer-BLAST was developed to help users make primers that are specific to the input PCR template. It uses Primer3 to design PCR primers and then submits them to BLAST...
Gel Layout:. Lane 1 - Hyperladder 1. Lanes 2-6 = 5′ RACE Library. Lane 2 - nGSP1 (5′ RACE primer). Lane 3 - nGSP2 (3′ RACE primer). Lane 4 - Neg. Control (no RACE primers). Lane 5 - Neg. Control (nGSP1, no Universal primer). Lane 6 - Neg. Control (nGSP2, no Universal primer). Lane 7 - Empty. Lanes 8-12 = 3′ RACE Library. Lane 8 - nGSP1 (5′ RACE primer). Lane 9 - nGSP2 (3′ RACE primer). Lane 10 - Neg. Control (no RACE primers). Lane 11 - Neg. Control (nGSP1, no Universal primer). Lane 12 - Neg. Control (nGSP2, no Universal primer). First of all, we see the appropriate response of each primer only producing amplicons in their respective libraries (i.e. 5′ primer only works in 5′ RACE library). This simply confirms that the primers were designed correctly. Secondly, our negative controls are clean. Thirdly, we get distinct bands from both primers. The bands marked with blue arrows in the image above were excised and purified using Ultrafree DA spin columns (Millipore). These ...
Methylated/Non-methylated Control DNA & Primer Set DNA ZD5101-2 Methylated/Non-methylated Control DNA & Primer Set DNA ZD5101-2
Pick PCR primers and hybridization oligos Version: EMBOSS:6.6.0.0 Standard (Mandatory) qualifiers: [-sequence] seqall The sequence from which to choose primers. The sequence must be presented 5 to 3 [-outfile] outfile [*.eprimer32] Whitehead primer3_core program output file Additional (Optional) qualifiers (* if not always prompted): -[no]primer toggle [Y] Tell Eprimer32 to pick primer(s) * -task menu [1] Tell Eprimer32 what task to perform. Legal values are 1: Pick PCR primers, 2: Pick forward primer only, 3: Pick reverse primer only, 4: No primers needed. (Values: 1 (Pick PCR primers); 2 (Pick forward primer only); 3 (Pick reverse primer only); 4 (No primers needed)) -hybridprobe toggle [N] An internal oligo is intended to be used as a hybridization probe (hyb probe) to detect the PCR product after amplification. * -mishyblibraryfile infile Similar to MISPRIMING-LIBRARY, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library ...
Picks PCR primers and hybridization oligos Version: EMBOSS:6.4.0.0 Standard (Mandatory) qualifiers: [-sequence] seqall The sequence from which to choose primers. The sequence must be presented 5 to 3 [-outfile] outfile [*.eprimer3] Whitehead primer3_core program output file Additional (Optional) qualifiers (* if not always prompted): -[no]primer toggle [Y] Tell EPrimer3 to pick primer(s) * -task menu [1] Tell EPrimer3 what task to perform. Legal values are 1: Pick PCR primers, 2: Pick forward primer only, 3: Pick reverse primer only, 4: No primers needed. (Values: 1 (Pick PCR primers); 2 (Pick forward primer only); 3 (Pick reverse primer only); 4 (No primers needed)) -hybridprobe toggle [N] An internal oligo is intended to be used as a hybridization probe (hyb probe) to detect the PCR product after amplification. * -mishyblibraryfile infile Similar to MISPRIMING-LIBRARY, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather ...
use Bio::PrimerDesigner; my $pd = Bio::PrimerDesigner-,new; # # Define the DNA sequence, etc. # my $dna = CGTGC...TTCGC; my $seqID = sequence 1; # # Define design parameters (native primer3 syntax) # my %params = ( PRIMER_NUM_RETURN =, 2, PRIMER_SEQUENCE_ID =, $seqID, SEQUENCE =, $dna, PRIMER_PRODUCT_SIZE =, 500-600 ); # # Or use input aliases # %param = ( num =, 2, id =, $seqID, seq =, $dna, sizerange =, 500-600 ); # # Design primers # my $results = $pd-,design( %params ) or die $pd-,error; # # Make sure the design was successful # if ( !$results-,left ) { die No primers found\n, $results-,raw_data; } # # Get results (single primer set) # my $left_primer = $results-,left; my $right_primer = $results-,right; my $left_tm = $results-,lefttm; # # Get results (multiple primer sets) # my @left_primers = $results-,left(1..3); my @right_primers = $results-,right(1..3); my @left_tms = $results-,lefttm(1..3 ...
This is the amplicon context sequence in accordance with the minimum information for the publication of real-time quantitative PCR experiements (MIQE) guidelines. For more details, please refer to the following publication, "Primer Sequence Disclosure: A Clarification of the MIQE Guidelines." ...
This is the amplicon context sequence in accordance with the minimum information for the publication of real-time quantitative PCR experiements (MIQE) guidelines. For more details, please refer to the following publication, "Primer Sequence Disclosure: A Clarification of the MIQE Guidelines." ...
Online resource for oligonucleotide and primer design, calculting melting temperature, secondary structure determination, PCR resource and primer databases
Any ideas of any tools that can help me with the above steps? For Primer design I have used eprimer3 thus far (with invariably satisfactory results) - but it seems to not support the concept of arbitrary degenerate primers.. Ideally, if I had a function which could sort out the first of the above criteria, I could use Biopython and BLAST to script the rest myself. Though the binding frequency would take hours if not a day or two, for BLAST to determine it. It would greatly help if anybody already wrote something more elaborate for this. ...
c. Next, is a detailed view of each primer sequence (query) aligned to the nucleotide sequence of the search hit (subject). Notice that amatch to the forward primer (nucleotides 1-42), and a match tothe reverse primer (nucleotides 44-68) are within the sameAccession. Also notice that position 43 of the forward primer ismissing. What does this mean? 3. Determine the predicted length of the product that the primer set would amplify in a PCR reaction (in vitro): a. In the list of significant alignments, notice the E-values in the column on the right. The Expectation or E-value is the number ofalignments with the query sequence that would be expected tooccur by chance in the database. The lower the E-value, the higherthe probability that the hit is related to the query. What does the E-value of 6e-12 mean? b. Note the names of any significant alignments that have E-values less than 0.1. Do they make sense? What do they have in common? c. Scroll down to the Alignments section to see exactly where the ...
primers. Consult the Table of Concentrations when you are ready to submit the primer for sequencing. If there are other primers you think we should consider stocking, please let us know. These should ...
How does this compare/differ to UDPP and/or TFSI? The Smashbox Lid Primer itself has pretty nice packaging in the sense that the wand is long, the tube is straight, so you are utilizing all the product in the tube without wasting it or having to cut it open like UDPP (Urban Decay Primer Potion), where its clear how much product is left that WOULD have gone to waste if it were not sliced open. UDPP and TFSI seemed a bit more watery in comparison to the Smashbox primer, not saying it IS watery by any means, but standing next to the Smashbox Lid Primer (which is nice and thick), they both seem "thin" when it comes to consistency and feel. When it came to vibrancy/pigmentation and endurance of wear, Smashbox was STILL a winner as you can in the photos below. It clearly brought out the color of my eyeshadows as well as lasting longer in wear in comparison to UDPP and TFSI ...
No, the primer site locations of the markers in the Investigator IDplex Plus Kit have not been changed. Only minor changes have been introduced for some markers, e.g., to improve A addition to the PCR products ...
휴먼 EXT2 qPCR primer pairs, confirmed in positive organizations; screened the primer with high specificity and high sensitivity.
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qPCR assays for rapid, sensitive microbial and viral detection Save time spent designing primers and probes by using predesigned assay sets. The following assays have been tested on the cobas omni Utility Channel: C. difficile; Influenza A, B /RSV; HSV / VZV. Primers and probes are HPLC purified and verified by mass spectrometry, delivering batch-to-batch consistency to provide confidence in your data.
From figure 13, it can be seen that N1 DNA treated with NSEQ1-RS2 primers, show a very light DNA band. This suggests that some DNA from the part of interest is giving out signals. Furthermore, N3 DNA treated with NSEQ1-RS2 primers did not give any signal other than the primer itself. Neither N1 or N3 DNAs, treated with 5NSEQ-RS1 primers, gave any signals. These results suggest that the NSEQ1 primer is the problem in the NSEQ1-RS2 primer mix and we dont have enough data to determine which primer is the problem from the 5NSEQ-RS1 primer mix (results from figure 10, regarding the HBT treated with 5NSEQ/HBT-PCR-REV were inconclusive ...
SybGREEN qpcr Primers and Standards Tools for RT-PCR Application Guide Table of Contents Package Contents and Storage Conditions... 1 Introduction... 3 SybGREEN qpcr Primer Pairs... 3 Gene Specific qpcr
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Payment terms for oligos, primers and probes are net 30 days. We manufacture high quality oligos, primers and probes at low cost. Please contact our...
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a) Primers are very cheap. Why dont you just design ten primers with rare restriction sites in the tails, amplify your five fragments with them, then digest with the appropriate restriciton enzymes? Any vector your lab already has could be acceptable then ...
I am researching the use of an inorganic zinc primer to protect hot spots on a unit. I did a bit of research into the chemistry behind it but was only able to c
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Yes, the layout of the QuantiTect Primer Assay Plates can be seen when ordering online via GeneGlobe. However, the drag-and-drop function for manual re-positioning of individual assays on the plate is only available for the 96-well format.. ...
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Is COM dead? To some, yes. For those who are just starting out with Win32 DNA programming however, it's a different story. In this article Nevile gives us a quick COM primer.
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All PrimeTime® qPCR Assay primers and probe are shipped dried down. The amount of primer and probe shipped depends on the assay scale ordered. The Mini scale is available only with probe containing 5 FAM™ dye, internal ZEN™ quencher, and 3 Iowa Black® FQ, and is sent at a fixed amount of 1 nmole primers (each) and 0.5 nmole probe. The Standard (500-20 µL reactions) and XL (2500-20 µL reactions) Assay scales have options for primer-to-probe ratio (ranging from 1:1 to 4:1), and a variety of dye/quencher combinations are available ...
DNA isolation was performed for the nine constructs that had not grown before. Plans were made for performing PCR with new primers tomorrow. ...
Did you think that sample prep and primer design was straight forward? What impact can the use of different housekeeping genes as controls have on your results? You will be shocked at what you find out. In this video, Sean Taylor of Bio-Rad Laboratories explains exactly what you must know to perform RT-qPCR according to…
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There are three choices of RT primer: oligo(dT), random primers, or a gene-specific primer. The priming strategy has a strong impact on the workflow applied, since each priming method has prerequisites and consequences.. Oligo(dT) priming is used to receive full-length copies of the mRNA. For cDNA library construction or cDNA labeling applications, oligo(dT) primers are almost always used to prime cDNA synthesis. Anchored oligo(dT)18 primers are designed to bind at the beginning of the poly(A) tail (rather than randomly within the often long tail) to generate full-length cDNAs. This avoids mispriming and unnecessary reverse transcription of the long poly(A) tail. Since the 5´ ends of long mRNAs are often underrepresented in cDNA mixtures, this primer is preferred for most applications.. However, if the message is long or does not have a poly(A) tail (as with prokaryotic mRNA), then random hexamer primers are used. Random hexamer primers bind throughout the entire length of RNA, ensuring reverse ...
Despite the many methodological options available for mutation detection, the choice is limited, by the necessity to ensure the highest sensitivity of detection for all types of changes while keeping costs and time of analysis within a reasonable limit. The present study extends to large and complex genes, like BRCA1, the applications of the FAMA method using large PCR amplicons (up to 1.4 kb). A key feature of this diagnostic strategy is the robust and economical two-step procedure for strand-specific labeling of PCR products using chimeric oligonucleotides and universal fluorescent primers (Fig. 1)⇓ . This labeling method is cost-effective, because it requires only one set of universal fluorescent primers, and allows reproducible amplification of all exons and splice sites of BRCA1 using homogeneous PCR conditions.. In the blind-test screening for changes in exon 11 (Table 2)⇓ different kinds of mutations and polymorphisms (various microdeletions/insertions involving 1 to 11 nucleotides as ...
Lasergenes PrimerSelect offers the best in advanced primer design software, enabling you to design and analyze primers for PCR, sequencing, probe hybridization and transcription.
Note: Optimal annealing temperatures are often higher than the Tm of the primers (approximately +5 to +10°C) and have to be determined empirically. For both primers, the Tm should be similar.. Bases that do not hybridize to the template may be added at the 5´ end of a primer (e.g., for introducing restriction sites into the amplification product). Primer concentrations between 0.1 and 0.6 µM are generally optimal. Higher primer concentrations may promote mispriming and accumulation of nonspecific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of desired product.. Note: For some systems, a higher primer concentration (up to 1 µM) may improve results. When testing new primers, always include a positive control reaction with a template that has been tested for function in PCR. This control shows whether the primers are working. The Human Genomic DNA from Roche is a good control template for evaluation of human primer ...
You need to first build an initial protocol and then optimize it.. The most important to determine the conditions is the polymerase used. Some polymerases work at higher or lower temperatures and will work faster or slower. Find the default protocol from the polymerase seller and this will give you a good idea where to start.. The second most important is the primers used. Depending on the GC % in the primers and their length, the times or temperature of each step will change, or if you prefer will need to be Optimized. The length of the DNA fragments will also determine the length of the cycles. Most polymerase sellers will includes steps to optimize the reaction depending on the quantity of DNA used, primer length and other parameters. For more information: Designing primers: Taq DNA polymerase protocol example:. Optimizing PCR ...
Further examination of the map shows that all of the reverse primers start at about the same place within the promoter region (~299,000), and the length differences are primarily due to choice of the forward primer, in the 3 downstream region of the gene. Since we are mostly interested in maximizing the promoter region, well choose Primer Pair 3 for further work. To save the sequence of the PCR product, along with its gene annotation, mouse over the map for Primer Pair 3 to bring up the menu shown above, and click on GenBank view. The GenBank view will appear in a new tab or window ...
Further examination of the map shows that all of the reverse primers start at about the same place within the promoter region (~299,000), and the length differences are primarily due to choice of the forward primer, in the 3 downstream region of the gene. Since we are mostly interested in maximizing the promoter region, well choose Primer Pair 3 for further work. To save the sequence of the PCR product, along with its gene annotation, mouse over the map for Primer Pair 3 to bring up the menu shown above, and click on GenBank view. The GenBank view will appear in a new tab or window ...
Traditional PCR methods can be used for viral diagnosis, however they are required to be highly sensitivity, specific and reproducible for this use. These criteria are largely dependent on the complementarity of the target nucleic acid to the primers and probe used in the PCR reaction. The primers and probe are designed to bind to a highly conserved genomic region and maximise the match. The presence of a mismatch reduces the amplification efficiency and sensitivity. Unfortunately, viruses have high genetic variability which makes it substantially more challenging to locate regions that are conserved in all subtypes of a particular virus.. A recent paper, published in Scientific Reports, investigated the development of a simpler qPCR method that is able to tolerate the presence of mismatches. The authors describe a method that is mediated by a high-fidelity DNA polymerase and uses a primer and a fluorescent primer (called a HFman probe).. ...
DNASTAR is a global software company offering the best in sequence analysis software, next-gen sequencing software, primer design software, and gene expression software.
To overcome some of the statistical uncertainties inherent in analyzing all four primer sets vs. the pooled data, we also aggregated the four separate datasets and compared this aggregate to the pooled data. (Essentially this amounts to averaging the four individual primer sets results and comparing this average to the pooled results). The results are shown in Figure 4. Again the nonparametric version gives higher results, but the confidence intervals overlap considerably. The most optimistic interpretation of Figure 4 is that, on average, the four primer sets can be expected to recover at most 60% of the total microbial diversity recoverable using the pooled approach.. The above statistical analyses showed that estimates of the protistan richness of the sample based on single PCR primer data sets do not significantly differ, and varied between 43 (SE 17) and 107 (SE 34) species (defined as OTUs grouping sequences that share at least 99% identity) (Figure 2). These analyses also showed that ...
High Resolution Melting (HRM) Analysis; is a powerful technology for SNP genotyping, mutation scanning and sequence scanning in DNA samples. Learn how HRM technology works, how HRM primers are designed, advantages and applications of HRM
High specificity and sensitivity Mouse, Adiponectin qPCR primer pair is designed by proprietary primer design algorithm and validated by strict process. Quote for bulk production.
bad_fwdprimers = [bad_fwdprimers_gc, bad_fwdprimers_tm,... bad_fwdprimers_dimers, bad_fwdprimers_hairpin,... bad_fwdprimers_clamp, bad_fwdprimers_repeats]; bad_revprimers = [bad_revprimers_gc, bad_revprimers_tm,... bad_revprimers_dimers, bad_revprimers_hairpin,... bad_revprimers_clamp, bad_revprimers_repeats]; good_fwdpos = find(all(~bad_fwdprimers,2)); good_fwdprimers = fwdprimerlist(good_fwdpos,:); good_fwdprop = fwdprimerprops(good_fwdpos); N_good_fwdprimers = numel(good_fwdprop) good_revpos = find(all(~bad_revprimers,2)); good_revprimers = revprimerlist(good_revpos,:); good_revprop = revprimerprops(good_revpos); N_good_revprimers = numel(good_revprop) figure imagesc([bad_fwdprimers any(bad_fwdprimers,2)]); title(Filtering candidate forward primers); ylabel(Primer location); xlabel(Criteria); ax = gca; ax.XTickLabel = char({%GC,Tm,Dimers,Hairpin,GC clamp,Repeats,All}); ax.XTickLabelRotation = 45; colorbar ...
Prima selects oligonucleotide primers for a template DNA sequence. The primers may be useful for the polymerase chain reaction (PCR) or for DNA sequencing. You can allow Prima to choose primers from the whole template or limit the choices to a particular set of primers listed in a file. ...
We performed qRT-PCR reactions on RNA preparations. extracted from strain 2787 at different points during growth in LB broth at 37 °C with shaking. We used primers specific for the aah gene, for the aidA gene and a pair of primers amplifying a region encompassing the 3′-end of aah and the 5′-end of aidA (Fig. 1a). Primers specific for the rpoD genes were used to normalize Pembrolizumab and compare the amounts of transcripts that could be amplified (Fig. 2a). The amplification with the aah-aidA primers shows that the two genes can be transcribed from a single bicistronic message. The levels of mRNA detected with the three pairs of primers varied significantly during growth. The pattern of variation was similar for the three primer pairs: there was an initial decrease during the log phase, most likely because of dilutions of existing. RNA pools from the overnight culture, and then an abrupt increase in the early-stationary phase. This has been observed with RpoS-controlled genes (Gordia & ...
Issuu is a digital publishing platform that makes it simple to publish magazines, catalogs, newspapers, books, and more online. Easily share your publications and get them in front of Issuus millions of monthly readers. Title: Primer Volume 24, Author: Primer Media, Inc., Name: Primer Volume 24, Length: 36 pages, Page: 1, Published: 2010-03-01
Human CTNNA2 qPCR primer pairs, confirmed in positive organizations; screened the primer with high specificity and high sensitivity.
This database allows retrieval of precomputed tandem repeats from the selected genome. It also allows to easily design primers for amplification of a DNA fragment containing the tandem repeat. The primers may be used for PCR simulation with a specific genome or with all genomes within the selected genera ...
This database allows retrieval of precomputed tandem repeats from the selected genome. It also allows to easily design primers for amplification of a DNA fragment containing the tandem repeat. The primers may be used for PCR simulation with a specific genome or with all genomes within the selected genera ...
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TY - JOUR. T1 - Efficiency of reduced primer selectivity and bulked DNA analysis for the rapid detection of AFLP polymorphisms in a range of crop species. AU - van Treuren, R.. PY - 2001. Y1 - 2001. U2 - 10.1023/A:1004003121622. DO - 10.1023/A:1004003121622. M3 - Article. VL - 117. SP - 27. EP - 37. JO - Euphytica. JF - Euphytica. SN - 0014-2336. ER - ...
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i just took my primer off due to leaks, my question is how do u prime it w/o the primer? i heard someone says to blow the pressure line til the fuel
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I´m always on the hunt for something that can help my pores appear smaller, and I have a box full of different primers. Most of them are ok, and probably more than ok as well, but I just don´t think I will ever get satisfied with anything unless my pores go away completely:p So I might not be the most fair person to judge a pore minimizing product, because we all know a primer won´t make any pores in the world dissapear completely. But it can make the problem less noticeable, there and then - so let´s get into my thoughts, is it worth the hype or not ...
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PROMER performs both functions of primer and probe in a single nucleic acid. As a result, PROMER requires smaller amount of oligos, reduces dimer formation, and is more cost-effective. ...
BioMed Research International is a peer-reviewed, Open Access journal that publishes original research articles, review articles, and clinical studies covering a wide range of subjects in life sciences and medicine. The journal is divided into 55 subject-specific sections.
Chlamydiae are implicated in a variety of clinically and economically important diseases in livestock and companion animals. These bacteria are associated with abortion, conjunctivitis, encephalomyelitis, enteritis, pneumonia, and polyarthritis in ruminants. Infection with these bacteria is the most common cause of abortion in sheep and goats and also causes zoonotic infection in humans which, in pregnant women, can result in spontaneous abortion.
The goals of this investigation were to identify and evaluate the use of polymorphic microsatellite marker (PMM) analysis for molecular typing of seventeen plant pathogenic fungi. Primers for di-, tri-, and tetranucleotide loci were designed directly from the recently published genomic sequence of Mycospherlla graminicola and Fusarium graminearum. A total of 20 new microsatellite primers as easy-to-score markers were developed. Microsatellite primer PCR (MP-PCR) yielded highly reproducible and complex genomic fingerprints, with several bands ranging in size from 200 to 3000 bp. Of the 20 primers tested, only (TAGG)4, (TCC)5 and (CA)7T produced a high number of polymorphic bands from either F. graminearum or F. culmorum. (ATG)5 led to successful amplifications in M. graminicola isolates collected from Germany. Percentage of polymorphic bands among Fusarium species ranged from 9 to 100%. Cluster analysis of banding patterns of the isolates corresponded well to the established species delineations based
Today, there are several algorithms and tools available for aligning nucleotide sequences locally. In this master thesis is especially MegaBlast, bu also other tools in the BLAST family, used in the construction of two different applications. What these applications have in common, is that they aim to find all perfect matches when aligning short nucleotide sequences to a large nucleotide database. Both accuracy and time efficiency is emphasized in the applications. UniquePrimers, one of the applications, is constructed in the context of verifying potential primers for a polymerase chain reactions. The application uses the primers as input, and returns all sequences that include the primers within a specified distance between the primers. This means that the application verifies whether or not a pair of primers are unique for only one specific database sequence. A search takes approximately from 15 seconds up to 5 minutes to execute. By using MegaBlast and word length (W) 12, it is guaranteed ...
After you are done, you want to either coat with epoxy primer or do any filler work. With the wet coat system we used an epoxy primer under polyester paint. This sale is for a gallon of fast dry grey 2. Looks like I could have skipped wetsanding the epoxy primer with 400 grit but oh well, at the time I decided Id rather do it right than doing it twice If I had skipped wetsanding the epoxy primer, how much time would I need to wait before I layed down the catalyst primer over the freshly layed epoxy primer ? BTW thanks for the info. Klass Kote Epoxy Auto Primer is a high build primer sealer with excellent adhesion and corrosion resistance. Dry paint (check drying temperature and time) 5. SURFACE PREPARATIONS Clean surface with a Wax and Grease Remover. Although a bit more complicated to use than the spray epoxy primer, this type is the more popular one. The Spray Max 2K Epoxy Rust Cure Primer is an efficient aerosol primer that shows properties of exceptional adhesion, corrosion resistance, and ...
A primer binding site is a region of a nucleotide sequence where an RNA or DNA single-stranded primer binds to start replication. The primer binding site is on one of the two complementary strands of a double-stranded nucleotide polymer, in the strand which is to be copied, or is within a single-stranded nucleotide polymer sequence. A PCR primer binding site is a site where a polymerase chain reaction (PCR) primer binds, to prime duplication of a complement to an existing DNA or RNA sequence. The HIV primer binding site is a structured RNA element in the genomes of retroviruses to which tRNA binds to initiate reverse transcription. In HIV, the tRNA is tRNA(3)(Lys) although it can use other tRNAs. It consists of 18 nucleotides and follows the U5 region of the 5-long terminal repeat (LTR) of the retrovirus. Berg, Jeremy M.; Tymoczko, John L. & Stryer, Lubert. (c. 2002). "DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites". Biochemistry. New York: W. H. Freeman and Co. ...
Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously (as if performing many separate PCR reactions all together in one reaction). This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR. Multiplex-PCR was first described in 1988 as a method to detect deletions in the dystrophin gene. It has also been used with the steroid sulfatase gene. In 2008, multiplex-PCR was used for analysis of microsatellites and SNPs. Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple sequences at once, additional information may be gained from a single test run that otherwise would ...
As a first step in developing a quick, accurate and simple method for the diagnosis of red ring disease, the loop-mediated isothermal amplification (LAMP)-based identification procedure was applied to the causative agent, Bursaphelenchus cocophilus. Two LAMP primer sets were designed using two loci of ribosomal RNA genes, i.e., D2-D3 expansion segments of the large subunit (D2-D3 LSU), and internal transcribed spacers (ITS). Within those two sets of primers, the D2-D3 LSU primer set successfully yielded amplicons from B. cocophilus nematode lysate prepared from 3-year-old DESS-fixed specimens. The specificity of the primers was examined using 18 species of confamilial Aphelenchoididae nematodes and primer sensitivity was tested using a diluted series of B. cocophilus lysate. The primer set did not amplify the DNA from other aphelenchoidids, and sensitivity was achieved by 1:100 diluted B. cocophilus DNA (roughly 1/1500 of total DNA from a single third-stage juvenile).
This study was designed to optimize and apply the use of loop-mediated isothermal amplification (LAMP) as an alternative to conventional polymerase chain reaction (PCR) for the detection of herpesvirus of turkeys (HVT) (FC 126 strain) in vaccinated and non-vaccinated poultry in Nigeria
Rapid and Sensitive Detection of Salmonella spp. by Using a Loop-Mediated Isothermal Amplification Assay in Duck Carcass Sample - loop-mediated isothermal amplification;Salmonella spp.;screening;duck;
Since presently-available DNA sequencing technologies are ill-suited for reading long sequences, large pieces of DNA (such as ... Wooley, JC; Godzik, A; Friedberg, I (Feb 26, 2010). "A primer on metagenomics". PLoS Comput Biol. 6 (2): e1000667. doi:10.1371/ ... Sequence assembly refers to the reconstruction of a DNA sequence by aligning and merging small DNA fragments. It is an integral ... In bioinformatics, sequence analysis is the process of subjecting a DNA, RNA or peptide sequence to any of a wide range of ...
"Primers, 16S ribosomal DNA - François Lutzoni's Lab". lutzonilab.net. Arquivado dende o orixinal o 2012-12-27.. ... O par cebador ou primer máis común foi concibido por Weisburg et al.[5] e denomínase actualmente 27F e 1492R; porén, para ... James, Greg (15 May 2018). "Universal Bacterial Identification by PCR and DNA Sequencing of 16S rRNA Gene". PCR for Clinical ... Kolbert CP, Persing DH (June 1999). "Ribosomal DNA sequencing as a tool for identification of bacterial pathogens". Current ...
The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to ... This is particularly useful when testing suitable annealing temperatures for PCR primers. ... Later, the PCR process was adapted to the use of thermostable DNA polymerase from Thermus aquaticus, which greatly simplified ... Weier, HU; Gray, JW (Jul-Aug 1988). "A programmable system to perform the polymerase chain reaction". DNA (Mary Ann Liebert, ...
Finally, the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA. By using two ... for rapid detection of viral genomic DNA or RNA, pathogenic bacterial genomic DNA, as well as short length aptamer DNA. The ... "Rapid PCR". Dna.utah.edu. Retrieved 2014-06-21. "PCR primers work using standard RPA reagents". TwistDx. Retrieved 2015-10-19 ... Recombinases are capable of pairing oligonucleotide primers with homologous sequence in duplex DNA. SSB bind to displaced ...
... end of an existing DNA molecule. Terminal transferase is one of the few DNA polymerases that can function without an RNA primer ... a DNA methyltransferase is a transferase that catalyzes the transfer of a methyl group to a DNA acceptor. In practice, many ... Terminal transferases are transferases that can be used to label DNA or to produce plasmid vectors. It accomplishes both of ... In the EC system of classification, the accepted name for RNA Polymerase is DNA-directed RNA polymerase. Described primarily ...
Universal matK primers can be used for DNA barcoding of angiosperms. LtrA, an open reading frame found in the Lactococcus ... Yu J, Xue J, Zhou S (2011). "New universal matK primers for DNA barcoding angiosperms". Journal of Systematics and Evolution. ...
With this strategy, PCR primers specific to a variant region of DNA are used (called SSP-PCR). If a product of the right size ... a b Hurley CK (1997). "DNA-based typing of HLA for transplantation." In Leffell MS, Donnenberg AD, Rose NR, eds. (1997) ... To interpret this table, it is necessary to consider that an allele is a variant of the nucleotide (DNA) sequence at a locus, ... cellular assay is being replaced by DNA-based typing method.[20] ...
Primosomes gives RNA primers for DNA synthesis to strands. Notes[edit]. Phi X is regularly used as a positive control in DNA ... DNA pilot protein (or minor spike protein) J. 60 in virion. Binds to new single-stranded phage DNA; accompanies phage DNA into ... Nicks RF DNA to initiate rolling circle replication; ligates ends of linear phage DNA to form single-stranded circular DNA ... The phi X 174 (or ΦX174) bacteriophage is a single-stranded DNA (ssDNA) virus that infects Escherichia coli, and the first DNA- ...
Di sini, yang dimaksud adalah mengubah "teks" DNA menjadi RNA. Sebenarnya, yang berubah hanyalah basa nitrogen timina di DNA ... Hasil transkripsi yaitu berkas RNA yang masih "mentah" yang disebut mRNA primer.[7] Di dalamnya terdapat fragmen berkas untuk ... TAC pada berkas DNA). Berkas DNA yang ditempel oleh RNA polimerase disebut sebagai berkas templat, sementara berkas pasangannya ... Intensitas transkripsi ditentukan oleh keadaan di sejumlah bagian tertentu pada DNA. Ada bagian yang disebut suppressor yang ...
DNA polymorphisms amplified by arbitrary primers are useful genetic markers. Nucleic Acids Res.18,6531-6535 (1990). 21.Stumpf ... as well as on DNA-DNA hybridization assays and phenotypic characterizations. The type strain EN-119T was isolated from ... On the basis of DNA relatedness, both organisms could be included in a single taxon. However, the CDC enteric group 69 was ... decreased accumulation and DNA gyrase protection. J. Antimicrob. Chemother.51,1109-1117 (2003). 107.Perichon B, Courvalin P, ...
". "DNA report on RGF". "An Insight into the Rajiv Gandhi Foundation". "cambridge trust RGF Partnership". "Rajiv Gandhi UK ... ". "Beyond the Primer by Arvind gupta" (PDF). "Business Wire India article". libraries and librarianship in India. "Jagriti ...
SGI-DNA Gibson Assembly Cloning Guide. *Gibson Assembly Primer Design Tool. *Gibson Assembly Site Directed Mutagenesis Primer ... The DNA polymerase incorporates nucleotides to fill in any gaps.. *The DNA ligase covalently joins the DNA of adjacent segments ... It requires that the DNA fragments contain ~20-40 base pair overlap with adjacent DNA fragments. These DNA fragments are mixed ... This DNA assembly method has many advantages compared to conventional restriction enzyme/ligation cloning of recombinant DNA. ...
A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or ... the development and use of evolutionarily conserved sets of PCR primers. the use of microsatellite loci that vary among ... The marker could be a short DNA sequence, such as a sequence surrounding a single base-pair change, known as a single ... the development of advanced DNA sequencing techniques. Many things utilized for studying larger organisms has not been possible ...
"DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Markers". Nucleic Acids Research. 18 (22): 6531-6535. ... Books Kidwell, K. K.; Osborn, T. C. (1992). "Simple Plant DNA Isolation Procedures". In Beckman, J. S.; Osborn, T. C. Plant ... Doyle, J. J.; Doyle, J. L. (1987). "A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue". Phytochemical ... Sharma, R.; Mahla, R. H.; Mohapatra, T.; Bhargava, C. S.; Shama, M. M. (2003). "Isolating Plant Genomic DNA Without Liguid ...
"Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487-491. doi:10.1126/ ... DNA, based on A-T, C-G base pairs, are formed in well-aligned sequences. Through precise sequences of DNA, 20 amino acids are ... At first, taking DNA or RNA as templates, scientists developed a series of peptide nucleic acid (PNA)-based polymers, without ... In nature, DNA, RNA, proteins and other macromolecules can also be recognized as sequence-controlled polymers for their well- ...
of the domestic fowl using DNA polymorphisms amplified by arbitrary primers". Parasitology Research. 79 (2): 98-102. PMID ... DNA assays and recombinant DNA techniques. PCR has proven most useful for outbreak surveillance. Prior to these methods, ...
... more on AM fungal specific primers below) 2 μl template DNA PCR: Run on an MWG AG Biotech Primus 96 Plus thermocycler 15 ... "Kruger" primers yielded relatively higher diversity parameters than other comparable primers (LSU, ITS2) "Kruger" primers ... primer utilizes the partial SSU and the ITS. Currently, there is no consensus as to which primers or primer sets, being used ... and repeatable primers or primer sets. Currently there are four common AM fungal specific markers/primers used in genetic ...
These tools are used to optimize the design of primers for target DNA or cDNA sequences. Primer optimization has two goals: ... The last 10-12 bases at the 3' end of a primer are sensitive to initiation of polymerase extension and general primer stability ... On the other hand, FastPCR, a commercial application, allows simultaneous testing of a single primer or a set of primers ... nor should the primer pairs bind to conserved regions of a gene family. If the selectivity is poor, a set of primers will ...
The outer primers (F3 and B3) anneal to the template strand and also generate new DNA. These primers are accompanied by DNA ... The F3 primer, with DNA polymerase, binds to this end and generates a new double stranded DNA molecule while displacing the ... Out of the 4 primers involved, two of them are "inner primers" (FIP and BIP) which are designed to synthesize new DNA strands. ... The B3 primer binds to this side of the template strand as well, and with the help of DNA polymerase simultaneously creates a ...
The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to ... This is particularly useful when testing suitable annealing temperatures for PCR primers. Weier, HU; Gray, JW (Jul-Aug 1988). " ... The earliest thermal cyclers were designed for use with the Klenow fragment of DNA polymerase I. Since this enzyme is destroyed ... Later, the PCR process was adapted to the use of thermostable DNA polymerase from Thermus aquaticus, which greatly simplified ...
The target DNA undergoes the first run of polymerase chain reaction with the first set of primers, shown in green. The ... Conventional PCR requires primers complementary to the termini of the target DNA. The amount of product from the PCR increases ... A commonly occurring problem is primers binding to incorrect regions of the DNA, giving unexpected products. This problem ... Polymerase chain reaction itself is the process used to amplify DNA samples, via a temperature-mediated DNA polymerase. The ...
The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a ... ISBN 0-07-284846-4. Muse, Spencer V.; Gibson, Greg (2004). A primer of genome science. Sunderland, Mass: Sinauer Associates. ... Use the enzyme DNA ligase to seal the DNA fragments into the vector. This creates a large pool of recombinant molecules. These ... Extract and purify DNA. Digest the DNA with a restriction enzyme. This creates fragments that are similar in size, each ...
DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting in a complex mixture of DNA ... PCR primer design, dedicated software exist for the purpose. Software for the purpose include for example ApE [1] (open source ... The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA ... Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is ...
"Blocking primers to enhance PCR amplification of rare sequences in mixed samples - a case study on prey DNA in Antarctic krill ... Primer selection is an important step, as anything that cannot be targeted by the primer will not be amplified and thus will ... it was estimated that the total global environmental DNA sequencing effort had produced less than 1 percent of the total DNA ... "A Primer on Metagenomics". PLoS Computational Biology. 6 (2): e1000667. Bibcode:2010PLSCB...6E0667W. doi:10.1371/journal.pcbi. ...
Uses gene specific degenerate primers to amplify pieces of DNA, these fragments are resolved using electrophoresis, and then ... DNA gyrase (topoisomerase II) subunit A (point mutation C7313T). SH1553. parC (grlA). Topoisomerase IV subunit A (point ... However, S. haemolyticus does have unique chromosome regions distributed near oriC (the origin of chromosomal DNA replication ... Biofilm formation is influenced by a variety of factors including carbohydrates, proteins, and extracellular DNA. Detachment ...
DNA motifs are specific short DNA sequences, often 8-20 nucleotides in length , which are statistically overrepresented in a ... This is done by ligating universal primers to all fragments. However, 5C has relatively low coverage. The 5C technique ... This takes place at low DNA concentrations in the presence of T4 DNA ligase, such that ligation between cross-linked ... In 1948, R. D. Hotchkiss discovered DNA methylation. In 1953, Watson and Crick discovered the double helix structure of DNA. In ...
Je odličen primer neoklasične arhitekture tega območja. Po odločilnem obdobju za Grčijo leta 1912-1922 je Pirej doživel ... pristanišče je bilo tudi dopolnjeno in posodobljeno s poglabljanjem dna, gradnjo Kraljevega pristanišča, Trumba Pier, in ...
Certain configurations have been shown to stabilize Watson-Crick antiparallel duplex DNA. TINA-DNA primers have been shown to ... stabilize Hoogsteen triplex DNA formation from double-stranded DNA (dsDNA) and TFOs. Its ability to twist around a triple bond ... Diagnostic assays using DNA hybridization are limited by the dissociation of antiparallell duplex helices. This can be improved ... Studies show that the greatest increase in stability occurred when intercalating primers were used at the 3' and 5' ends. ...
... primer; 0.2-4.0 ng/ul template DNA; and 0.02-0.06 U/ul (0.5-1.5 units) Taq DNA polymerase. PCR conditions. A Perkin-Elmer Cetus ... All primers were 10 bases in length. Primers were purchased from Operon Technologies, Alameda CA. Primers discussed in the text ... primers A01, B16, (2 markers); Sp B, primers C07, B02, C15 (4 markers); Sp C, primers A01, C16, C19, D01 (7 markers); and Sp D ... DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nuc Acids Res 18: 6531-6535.. Williams JGK, ...
... or by adding to the PCR reagents a single-stranded DNA binding protein which blocks reaction at temperatures below about 50 C. ... Because cellular preparations normally should contain little single-stranded DNA, the amount of primers in a reaction permits ... and 6 μM of each primer. For "single primer pair" experiments, the primers were PV 1 and PV2, dictating a 449 bp product from ... "Single-stranded DNA binding protein" (SSB) refers to a polypeptide which binds to single-stranded DNA more tightly than to ...
PCR amplification of megabase DNA with tagged random primers (T-PCR).. Grothues D1, Cantor CR, Smith CL. ...
The present invention provides these E. suis primers and a method to use these primers in a PCR protocol to provide a highly ... suis specific primers in PCR with DNA from swine blood increases the sensitivity of current DNA hybridization protocols for ... These primers are capable of effectively hybridizing and serving as primers for the thermostable DNA polymerase used in the ... Saiki, Randall K., Primer Directed Enzymatic Amplification of DNA With a Thermostable DNA Polymerase, Science 239:487 491 (1988 ...
Template and primer requirements for DNA Pol θ-mediated end joining Message Subject (Your Name) has sent you a message from ... Template and primer requirements for DNA Pol θ-mediated end joining. Peng He and Wei Yang ... Polθ requires a minimum of 2 bp and optimally 4 bp between a template/primer pair for efficient and processive DNA synthesis. ... DNA Pol θ shares structural homology with the high-fidelity replicases, and its polymerase domain (Polθ) has been shown to ...
... region are shown in the Forward primer and Reverse Primer boxes.. In the Primer Parameters section, we set the Minimum PCR ... those primers are. Therefore, you need to make a special note of the primer data from the Primer-BLAST results.. ... see the sequences of the primers, as well as other data on the PCR primers. For comparison, results for Primer pairs 2 and 3 ... How does Primer-BLAST work?. The search for primers is essentially a 2-step process:. 1. Use the Primer3 program to design ...
... including random primers and hexamers, oligo (dT) primers, and sequencing primers. These products are commonly used in a ... Learn about the different pre-synthesized DNA primers and oligos that are available, ... pre-designed DNA primers and oligos. These products include random primers, Oligo (dT) primers, gene specific primers for ... Sequencing primers. Short RNA or DNA oligonucleotides are often used as primers in PCR reactions to amplify a specific target ...
In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete ... Conclusions/Significance Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis ... These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete ... Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics ...
Big peaks for a short time, then nothing - too much DNA.. Nothing - bad primer, loss of DNA, bad cycling conditions.. If your ... Plasmid DNA Sequencing with Universal Primers - (Mar/08/2007 ). Hi all, Ive been having some trouble with sequencing. The gene ... The next important thing the amount of DNA and Big Dye terminator in used. The amount of DNA I use is about 400- 500ng.. ... The DNA has to be the cleanest you can possibly get it to be. No RNA , no proteins, removal of phenol must be complete, if your ...
... vijay at ust.hk vijay at ust.hk Mon Sep 2 04:29:48 EST 2002 *Next ... Would anyone please suggest me the three primers for the TAIL reactions or the protocol from their own lab. Here is the vector ... student working now to clone a T-DNA insertion mutant of Cis-cinnamic acid.The mutant is isolated from Ecker and Alonso ... insertional line CS75100.I would like to design the primers for both Left and Right borders of the vector pROK2 for TAIL-PCR. ...
Our custom DNA oligos are made to your specifications, with rigorous quality control and quick turnaround. They can be used in ...
Molecular basis for stool-based DNA tests for colorectal cancer: a primer for clinicians.. Boland CR1. ... Tumor cells and DNA are shed into the fecal stream, and uniquely mutated genes can be identified that would indicate the likely ...
These primers are going to be the death of me. Im using MethPrimer to design them, but the primer dimer problem is endless, ... Is there ever a point where you just deal with primer dimers? I saw some posts about formamide but wasnt sure if it applied. ... posted in DNA Methylation and Epigenetics: designing MSP for a USF site in my gene of interest. ... Primer dimmer is a big issue for MSP or any bisulfite based PCR because the low sequence complexity of the modified DNA. You ...
... relating to PCR-DNA amlification it refers to the DNA primer. First of all why is this the case - DNA relpication:RNA primer, ... No DNA polymerase can. But they could start with DNA primers if such thing was available (and they do when repairing DNA). When ... DNA primer? Secondly when I look at the definition for a primer - it states that it is an RNA stand...so whats with the DNA ... RNA primers could be used but they are unstable and Rnases are frequent and really hard to get rid of, while DNA is easy to ...
This page illustrates high molecular weight calf thymus DNA in a liquid crystalline phase viewed under a combination of crossed ... Liquid Crystalline DNA. DNA undergoes a series of liquid crystalline phase transitions when it is compressed in vivo into ... DNA is a very unusual molecule that is shaped like a very long piece of string. The diameter of the DNA molecule is about 25-30 ... In addition, it is important to the cell to be able to access the DNA molecule for the purposes of genetic control, routine ...
This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the ... specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this ... Chiu, Angela Chen-Yen. DNA Typing of HLA-B by PCR with Primer Mixes Utilizing Sequence-Specific Primers, thesis, August 1997; ... DNA Typing of HLA-B by PCR with Primer Mixes Utilizing Sequence-Specific Primers ...
BcaBEST DNA Labeling Kit. The BcaBEST DNA Labeling Kit is a novel random primer labeling system that uses longer primers (9- ... Unlike E. coli Klenow fragment or modified T7 DNA polymerase, Bca polymerase allows stable polymerization of DNA even for ... Lambda DNA digested with Hind III, 25 ng/µL). Note. The kit name has been changed from "Ladderman Labeling Kit" to "BcaBEST ... In general, probes with specific activities of ,109 dpm/mg DNA obtained with [32P]-alpha-dCTP (~3,000 Ci/mmol, 111 TBq/mmol). ...
Here we developed a new set of 100 primer pairs optimized for amplification in Monocotyledons. Primer pairs amplify coding ( ... we demonstrated that this new set of primers is of great interest for population genetics and we anticipate that it will also ... Chloroplast DNA sequences are of great interest for population genetics and phylogenetic studies. However, only a small set of ... Chloroplast DNA Is the Subject Area "Chloroplast DNA" applicable to this article? Yes. No. ...
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. By RK Saiki, DH Gelfand, S Stoffel, SJ ... Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. By RK Saiki, DH Gelfand, S Stoffel, SJ ... Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase Message Subject. (Your Name) has forwarded a ... A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, ...
Home/ Forums/ DNA (PCR, Real Time PCR): Primers and Oligonucleotides. DNA (PCR, Real Time PCR): Primers and Oligonucleotides. ... PCR primers for mycoplasma 16S rRNA gene? (Page: 1, 2, 3, 4) by Jason King » Fri, 11/20/2009 - 12:30 194. by Rahul Date. Sat, ... Exon exon junction primers for RT-PCR by Haritha » Sat, 08/17/2013 - 02:39 2. by elhamzeinali. Wed, 02/10/2016 - 06:19 ... Long Primers not working? by Crystal Ramirez » Tue, 06/14/2016 - 11:51 8. by Ivan Delgado. Thu, 06/16/2016 - 13:10 ...
Primer-BLAST. You are here. Home » Products » Sequencher » Sequencher Features » Connections » Primer-BLAST ... Your new primers are automatically tagged with a GenBank style feature key that makes them easier to see in an alignment. The ... Use Primer-BLAST to analyze sequences and check specificity.. Parallelize your analyses - use several different sets of ... If you want to learn more about Primer-BLAST, check out the Sequencher Connections tutorial. ...
A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary ... Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.). ... Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV: DNA polymorphisms amplified by arbitrary primers are useful as ... A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary ...
OH with a photolabile protecting group which can temporarily cease DNA strand growth and can smoothly reinitia ... Photo-cleavable nucleotides for primer free enzyme mediated DNA synthesis A. S. Mathews, H. Yang and C. Montemagno, Org. Biomol ... The synthesised nucleotides are employed for terminating and reinitiating template-less DNA synthesis, using primer independent ... The use of this photolabile nucleotide in one step stop-start DNA synthesis is a novel strategy towards the precise assembly of ...
Mitochondrial DNA Sequencing Primers mtDNA sequencing is useful in evolutionary studies, single nucleotide polymorphism (SNP) ... within the D-loop region of human mitochondrial DNA (mtDNA). ...
How should I store my targeted DNA sequencing library prep assay primers?. Primer pools are stable for up to one year at -20 °C ...
  • You can Read Online Enzymology Primer For Recombinant Dna Technology here in PDF, EPUB, Mobi or Docx formats. (nwcbooks.com)
  • Using the four primer pairs in this set for real-time PCR, relative quantification of mtDNA is determined from the difference in Ct values for mtDNA and nDNA. (takarabio.com)
  • The absolute detection limit of the real-time PCR method was 0.025 ng DNA, and the relative detection limit was 0.002% (w/w) of positive samples. (nature.com)
  • In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxynucleotides. (rcsb.org)
  • DNA polymerases are the enzymes that replicate DNA in living cells. (scienceprimer.com)
  • DNA polymerases can not create new strands of DNA. (scienceprimer.com)
  • According to the National Center for Biotechnology Information, the primary role of DNA polymerases is to replicate the DNA of an organism, accurately and efficiently, during cell division. (reference.com)
  • The National Center for Biotechnology Information explains that there are many different types of DNA polymerases. (reference.com)
  • Without curation, commonly-used qPCR primer pairs yielded an extreme range of reaction efficiencies (25.9-100.1%), but when conditions were optimized, MFQPCR produced copy-number estimates comparable to traditional qPCR. (frontiersin.org)
  • The use of E. suis specific primers in PCR with DNA from swine blood increases the sensitivity of current DNA hybridization protocols for determining whether swine are infected with E. suis prior to the development of any clinical symptoms. (google.com)
  • Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. (asm.org)
  • An equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. (nih.gov)
  • This technique is based on the specific hybridization of a probe, which was designed for a certain species, with the DNA in the samples to be analyzed. (nature.com)
  • Primer Probe Hybridization: There is always the possibility of the primer hybridizing to the probe element, this will lead to linearization of the probe in an amplification-independent manner causing significant, target-independent fluorescence. (biosyn.com)
  • Would anyone please suggest me the three primers for the TAIL reactions or the protocol from their own lab. (bio.net)
  • Then you perform two primer extension reactions, one with radiolabelled dCTP (ddCTP) and another with dTTP (ddTTP). (methods.info)
  • Prepare two reactions containing the same amount of PCR product, 1xPCR buffer, 1 pM of the primer, 1 U Taq polymerease and 1uCi of 32 P dCTP or dTTP in 20 ul final volume. (methods.info)
  • Reactions are run on an ABI 3730XL capillary-based DNA Sequencer. (albany.edu)
  • An animated primer of 75 experiments that made modern genetics. (dnaftb.org)
  • Given how complex genetics can be, DNA ancestry tests today should mostly be done for curiosity's sake rather than for definitive results. (healthcentral.com)
  • Though it it is possible to use other tools, NEB recommends using our free tool, NEBuilder Assembly Tool, because it can design primers that consider the vector ends generated by a restriction enzyme digestion. (neb.com)
  • Here we present 10 high resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment variants complexed with deoxy-, ribo-, and dideoxynucleotides and a DNA substrate. (rcsb.org)
  • In prokaryotes, the particular form of the enzyme is called DNA polymerase III. (encyclopedia.com)
  • Bisulfite (BS) conversion-based and methylation-sensitive restriction enzyme (MSRE)-based PCR methods have been the most commonly used techniques for locus-specific DNA methylation analysis. (biomedcentral.com)
  • Bisulfite-based methods and methylation-sensitive restriction enzyme-based PCR (MSRE-PCR) methods have been widely used for detection of DNA methylation. (biomedcentral.com)
  • Interestingly, the polymerase used in the PCR is not a normal one, it is a special type of enzyme that is temperature stable, called Taq DNA polymerase . (geneticeducation.co.in)
  • To overcome this limitation, other methods aim to reduce the formation of PDs only, including primer design, and use of different PCR enzyme systems or reagents. (wikipedia.org)
  • rather, it is extended in situ to generate duplex linear DNA. (asm.org)
  • Welsh J, McClelland M: Fingerprinting genomes using PCR with arbitrary primers. (springer.com)
  • Hepadnaviruses replicate their circular DNA genomes via reverse transcription of an RNA intermediate. (asm.org)
  • The genomes of bacterial cells ( prokaryotes ), which lack a nucleus, are typically circular DNA molecules that associate with special structures in the cell membrane. (encyclopedia.com)
  • Conclusion: The development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. (byu.edu)
  • Designing specific and optimized primers for target regions is the most critical and challenging step in obtaining the adequate DNA methylation results using PCR-based methods. (biomedcentral.com)
  • Currently, no integrated, optimized, and high-throughput methylation-specific primer design software methods are available for both BS- and MSRE-based methods. (biomedcentral.com)
  • Therefore an integrated, powerful, and easy-to-use methylation-specific primer design pipeline with great accuracy and success rate will be very useful. (biomedcentral.com)
  • Detection of DNA in specimens comprising body fluids of tissues can be difficult because of the small quantity of DNA present or because of the presence in the specimen of other interfering materials, including DNA from a different source. (google.com)
  • Diagnosis of new world leishmaniasis: specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA. (ajtmh.org)
  • Therefore, if we want a larger PCR product that includes the flanking regions, we have to choose a larger region to be used for primer design. (umanitoba.ca)
  • student working now to clone a T-DNA insertion mutant of Cis-cinnamic acid.The mutant is isolated from Ecker and Alonso insertional line CS75100.I would like to design the primers for both Left and Right borders of the vector pROK2 for TAIL-PCR. (bio.net)
  • I'm using MethPrimer to design them, but the primer dimer problem is endless, but With such a high content of A's and T's I'm drowning. (protocol-online.org)
  • You can design and try several sets of primers that cover the same region, and optimize your PCR conditions. (protocol-online.org)
  • Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly? (neb.com)
  • Tools which design "Gibson" primers may not allow the design of primers that take advantage of this ability of NEBuilder HiFi. (neb.com)
  • PCR Primer Design focuses on primer design, which is critical to both the efficiency and the accuracy of the PCR. (springer.com)
  • In PCR Primer Design, authors describe basic approaches for PCR primer design in addition to specialized methods. (springer.com)
  • In such cases, the prediction of the correct insertion site must include careful sequence analyses on the one hand and a paralog specific primer design for experimental confirmation of the prediction on the other hand. (biomedcentral.com)
  • A newly developed primer design tool that takes paralogous regions into account was developed to streamline the confirmation process for paralogs. (biomedcentral.com)
  • An automatic online primer design tool that incorporates experience from the in-house confirmation of T-DNA insertion lines has been made available. (biomedcentral.com)
  • We have covered an entire article on how to design DNA primers for PCR. (geneticeducation.co.in)
  • High throughput PCR primer design software. (jcvi.org)
  • Please note that this service does not include design of primer(s). (dnaseq.co.uk)
  • We encourage them to review the information in our resource pages around primer design if they are new to this step. (dnaseq.co.uk)
  • Ultimately one cause of poor sequencing results is indeed poor primer design, thus please consider your design carefully as the MRC PPU DNA Sequencing and Services facility cannot be held responsible for such problems. (dnaseq.co.uk)
  • Ideally the primers should have as little secondary structure as possible. (biosyn.com)
  • Templates should be added in appropriate quantities (see Template Concentrations chart below), with a total well volume including template, water, and primer of 5 μl/well. (albany.edu)
  • If using your own custom primers, please see Primer Concentrations below and include with template shipment. (albany.edu)
  • Recommended concentrations for templates and primers are shown below. (albany.edu)
  • By this technique, selective enrichment of a specific DNA sequence can be achieved by exponential amplification of the target sequence. (google.com)
  • We evaluated the Leishmania Viannia -specific primers B1-B2 to detect Leishmania in normal skin and peripheral blood monocytes of patients with active cutaneous leishmaniasis. (ajtmh.org)
  • We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. (uio.no)
  • Generally, RNases H are defined as ribonucleotide-specific endonucleases, cleaving the RNA portion of RNA-DNA/DNA or RNA/DNA duplexes. (asm.org)
  • Upon a user request, T2 plants of the respective line are grown and the insertion site prediction is confirmed at GABI-Kat by PCR with an insertion site-specific primer and a T-DNA border primer, followed by sequencing of the amplicon. (biomedcentral.com)
  • If 99.9 percent of people have a G base in a specific DNA sequence and 0.1 percent have a C base, it's called a mutation. (dnadietplan.com)
  • The direct-to-consumer DNA ancestry test kits use a process called genotyping to find variants in specific areas of the DNA, which in turn can be linked to certain ancestral groups and ethnic regions, physical traits, and health conditions. (healthcentral.com)
  • With bad results from multiple trials of different primer sets, I'd be looking at template purity issues, though it's a tough call with out seeing the trace. (protocol-online.org)
  • Results: The developed primers successfully amplified DNA of their target organism from various soil DNA extracts. (byu.edu)
  • Taken together, these results show that BODIPY and BET direct DNA sequencing can accurately and precisely characterize complex mixed-base populations. (bcm.edu)
  • At the Morris family reunion of 2007 in Las Vegas, I revealed the results of our DNA test. (a3genealogy.com)
  • Using 1 primer will results in amplifying the original sequence in each cycle in a linear fashion: the primer will guide a single run of the polymerase. (stackexchange.com)
  • It's hard to turn on a TV today without seeing an ad hawking direct-to-consumer DNA ancestry tests, and I was as curious as anyone about what my results might be. (healthcentral.com)
  • It turns out that all three DNA ancestry test kits produced fairly uniform results: I found out I am largely Northern and Western European (Scandinavian, Finnish and Irish) and a quarter Ashkenazi Jewish. (healthcentral.com)
  • The bottom line: The DNA ancestry test kit results are best guesses (although my little experiment showed that the three companies I chose are all playing in the same ballpark). (healthcentral.com)
  • More tests will undoubtedly follow, and physicians are concerned about the accuracy of the testing, how to counsel patients about their DNA reports, and how well people will understand the results. (healthcentral.com)
  • While RNA is possible, it's much harder to synthesize (and keep stable) and DNA results in a purely DNA result that won't need to be replaced with DNA later. (stackexchange.com)
  • DNA sequencing results proved the utility of the designed primers. (biomedcentral.com)
  • The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so. (biomedcentral.com)
  • Is the Subject Area "Chloroplast DNA" applicable to this article? (plos.org)