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.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.Base Sequence: The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.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.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, 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 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.Real-Time Polymerase Chain Reaction: Methods used for detecting the amplified DNA products from the polymerase chain reaction as they accumulate instead of at the end of the reaction.RNA Polymerase II: A DNA-dependent RNA polymerase present in bacterial, plant, and animal cells. It functions in the nucleoplasmic structure and transcribes DNA into RNA. It has different requirements for cations and salt than RNA polymerase I and is strongly inhibited by alpha-amanitin. EC 2.7.7.6.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)DNA, Viral: Deoxyribonucleic acid that makes up the genetic material of viruses.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.DNA, Bacterial: Deoxyribonucleic acid that makes up the genetic material of bacteria.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).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.DNA-Directed RNA Polymerases: Enzymes that catalyze DNA template-directed extension of the 3'-end of an RNA strand one nucleotide at a time. They can initiate a chain de novo. In eukaryotes, three forms of the enzyme have been distinguished on the basis of sensitivity to alpha-amanitin, and the type of RNA synthesized. (From Enzyme Nomenclature, 1992).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.Genotype: The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS.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.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.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.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.Gene Expression: The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION.DNA Polymerase II: A DNA-dependent DNA polymerase characterized in E. coli and other lower organisms. It may be present in higher organisms and has an intrinsic molecular activity only 5% of that of DNA Polymerase I. This polymerase has 3'-5' exonuclease activity, is effective only on duplex DNA with gaps or single-strand ends of less than 100 nucleotides as template, and is inhibited by sulfhydryl reagents. EC 2.7.7.7.Multiplex Polymerase Chain Reaction: Methods for using more than one primer set in a polymerase chain reaction to amplify more than one segment of the target DNA sequence in a single reaction.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.DNA Polymerase III: A DNA-dependent DNA polymerase characterized in E. coli and other lower organisms but may be present in higher organisms. Use also for a more complex form of DNA polymerase III designated as DNA polymerase III* or pol III* which is 15 times more active biologically than DNA polymerase I in the synthesis of DNA. This polymerase has both 3'-5' and 5'-3' exonuclease activities, is inhibited by sulfhydryl reagents, and has the same template-primer dependence as pol II. EC 2.7.7.7.Sequence Analysis, DNA: A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis.DNA, Protozoan: Deoxyribonucleic acid that makes up the genetic material of protozoa.Electrophoresis, Agar Gel: Electrophoresis in which agar or agarose gel is used as the diffusion medium.Gene Amplification: A selective increase in the number of copies of a gene coding for a specific protein without a proportional increase in other genes. It occurs naturally via the excision of a copy of the repeating sequence from the chromosome and its extrachromosomal replication in a plasmid, or via the production of an RNA transcript of the entire repeating sequence of ribosomal RNA followed by the reverse transcription of the molecule to produce an additional copy of the original DNA sequence. Laboratory techniques have been introduced for inducing disproportional replication by unequal crossing over, uptake of DNA from lysed cells, or generation of extrachromosomal sequences from rolling circle replication.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, 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 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.Alleles: Variant forms of the same gene, occupying the same locus on homologous CHROMOSOMES, and governing the variants in production of the same gene product.RNA, Viral: Ribonucleic acid that makes up the genetic material of viruses.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.RNA Polymerase I: A DNA-dependent RNA polymerase present in bacterial, plant, and animal cells. The enzyme functions in the nucleolar structure and transcribes DNA into RNA. It has different requirements for cations and salts than RNA polymerase II and III and is not inhibited by alpha-amanitin. EC 2.7.7.6.RNA Polymerase III: A DNA-dependent RNA polymerase present in bacterial, plant, and animal cells. It functions in the nucleoplasmic structure where it transcribes DNA into RNA. It has specific requirements for cations and salt and has shown an intermediate sensitivity to alpha-amanitin in comparison to RNA polymerase I and II. EC 2.7.7.6.Immunohistochemistry: Histochemical localization of immunoreactive substances using labeled antibodies as reagents.Immunoglobulin Heavy Chains: The largest of polypeptide chains comprising immunoglobulins. They contain 450 to 600 amino acid residues per chain, and have molecular weights of 51-72 kDa.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.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.DNA Polymerase beta: A DNA repair enzyme that catalyzes DNA synthesis during base excision DNA repair. EC 2.7.7.7.Promoter Regions, Genetic: DNA sequences which are recognized (directly or indirectly) and bound by a DNA-dependent RNA polymerase during the initiation of transcription. Highly conserved sequences within the promoter include the Pribnow box in bacteria and the TATA BOX in eukaryotes.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.Cells, Cultured: Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.Exons: The parts of a transcript of a split GENE remaining after the INTRONS are removed. They are spliced together to become a MESSENGER RNA or other functional RNA.Restriction Mapping: Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.DNA, Neoplasm: DNA present in neoplastic tissue.Cell Line: Established cell cultures that have the potential to propagate indefinitely.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.DNA Mutational Analysis: Biochemical identification of mutational changes in a nucleotide sequence.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)In Situ Hybridization: A technique that localizes specific nucleic acid sequences within intact chromosomes, eukaryotic cells, or bacterial cells through the use of specific nucleic acid-labeled probes.Gene Expression Regulation: Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control (induction or repression) of gene action at the level of transcription or translation.Phylogeny: The relationships of groups of organisms as reflected by their genetic makeup.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)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.Cattle: Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor.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.Gene Expression Profiling: The determination of the pattern of genes expressed at the level of GENETIC TRANSCRIPTION, under specific circumstances or in a specific cell.Case-Control Studies: Studies which start with the identification of persons with a disease of interest and a control (comparison, referent) group without the disease. The relationship of an attribute to the disease is examined by comparing diseased and non-diseased persons with regard to the frequency or levels of the attribute in each group.Gene Frequency: The proportion of one particular in the total of all ALLELES for one genetic locus in a breeding POPULATION.Blotting, Western: Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.Time Factors: Elements of limited time intervals, contributing to particular results or situations.Point Mutation: A mutation caused by the substitution of one nucleotide for another. This results in the DNA molecule having a change in a single base pair.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.Gene Rearrangement: The ordered rearrangement of gene regions by DNA recombination such as that which occurs normally during development.Evaluation Studies as Topic: Studies determining the effectiveness or value of processes, personnel, and equipment, or the material on conducting such studies. For drugs and devices, CLINICAL TRIALS AS TOPIC; DRUG EVALUATION; and DRUG EVALUATION, PRECLINICAL are available.Kinetics: The rate dynamics in chemical or physical systems.Immunoglobulin Light Chains: Polypeptide chains, consisting of 211 to 217 amino acid residues and having a molecular weight of approximately 22 kDa. There are two major types of light chains, kappa and lambda. Two Ig light chains and two Ig heavy chains (IMMUNOGLOBULIN HEAVY CHAINS) make one immunoglobulin molecule.Polymorphism, Single-Stranded Conformational: Variation in a population's DNA sequence that is detected by determining alterations in the conformation of denatured DNA fragments. Denatured DNA fragments are allowed to renature under conditions that prevent the formation of double-stranded DNA and allow secondary structure to form in single stranded fragments. These fragments are then run through polyacrylamide gels to detect variations in the secondary structure that is manifested as an alteration in migration through the gels.Nucleic Acid Amplification Techniques: Laboratory techniques that involve the in-vitro synthesis of many copies of DNA or RNA from one original template.Enzyme-Linked Immunosorbent Assay: An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed.Paraffin Embedding: The infiltrating of tissue specimens with paraffin, as a supporting substance, to prepare for sectioning with a microtome.Feces: Excrement from the INTESTINES, containing unabsorbed solids, waste products, secretions, and BACTERIA of the DIGESTIVE SYSTEM.Blotting, Northern: Detection of RNA 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.Recombinant Proteins: Proteins prepared by recombinant DNA technology.Transcription Factors: Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process.Phenotype: The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment.Tumor Virus Infections: Infections produced by oncogenic viruses. The infections caused by DNA viruses are less numerous but more diverse than those caused by the RNA oncogenic viruses.RNA, Neoplasm: RNA present in neoplastic tissue.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 Replication: The process by which a DNA molecule is duplicated.Translocation, Genetic: A type of chromosome aberration characterized by CHROMOSOME BREAKAGE and transfer of the broken-off portion to another location, often to a different chromosome.RNA Replicase: An enzyme that catalyses RNA-template-directed extension of the 3'- end of an RNA strand by one nucleotide at a time, and can initiate a chain de novo. (Enzyme Nomenclature, 1992, p293)Papillomaviridae: A family of small, non-enveloped DNA viruses infecting birds and most mammals, especially humans. They are grouped into multiple genera, but the viruses are highly host-species specific and tissue-restricted. They are commonly divided into hundreds of papillomavirus "types", each with specific gene function and gene control regions, despite sequence homology. Human papillomaviruses are found in the genera ALPHAPAPILLOMAVIRUS; BETAPAPILLOMAVIRUS; GAMMAPAPILLOMAVIRUS; and MUPAPILLOMAVIRUS.Dog Diseases: Diseases of the domestic dog (Canis familiaris). This term does not include diseases of wild dogs, WOLVES; FOXES; and other Canidae for which the heading CARNIVORA is used.Genetic Variation: Genotypic differences observed among individuals in a population.Reproducibility of Results: The statistical reproducibility of measurements (often in a clinical context), including the testing of instrumentation or techniques to obtain reproducible results. The concept includes reproducibility of physiological measurements, which may be used to develop rules to assess probability or prognosis, or response to a stimulus; reproducibility of occurrence of a condition; and reproducibility of experimental results.Oligonucleotide Array Sequence Analysis: Hybridization of a nucleic acid sample to a very large set of OLIGONUCLEOTIDE PROBES, which have been attached individually in columns and rows to a solid support, to determine a BASE SEQUENCE, or to detect variations in a gene sequence, GENE EXPRESSION, or for GENE MAPPING.DNA-Binding Proteins: Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.Genes, Bacterial: The functional hereditary units of BACTERIA.Biopsy: Removal and pathologic examination of specimens in the form of small pieces of tissue from the living body.Electrophoresis, Polyacrylamide Gel: Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.Binding Sites: The parts of a macromolecule that directly participate in its specific combination with another molecule.Tumor Cells, Cultured: Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely.Bacterial Proteins: Proteins found in any species of bacterium.DNA, Fungal: Deoxyribonucleic acid that makes up the genetic material of fungi.Up-Regulation: A positive regulatory effect on physiological processes at the molecular, cellular, or systemic level. At the molecular level, the major regulatory sites include membrane receptors, genes (GENE EXPRESSION REGULATION), mRNAs (RNA, MESSENGER), and proteins.Molecular Diagnostic Techniques: MOLECULAR BIOLOGY techniques used in the diagnosis of disease.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)Plasmids: Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.Genetic Predisposition to Disease: A latent susceptibility to disease at the genetic level, which may be activated under certain conditions.Genetic Markers: A phenotypically recognizable genetic trait which can be used to identify a genetic locus, a linkage group, or a recombination event.Poly(ADP-ribose) Polymerases: Enzymes that catalyze the transfer of multiple ADP-RIBOSE groups from nicotinamide-adenine dinucleotide (NAD) onto protein targets, thus building up a linear or branched homopolymer of repeating ADP-ribose units i.e., POLY ADENOSINE DIPHOSPHATE RIBOSE.Viral Proteins: Proteins found in any species of virus.Protein Binding: The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.Cattle Diseases: Diseases of domestic cattle of the genus Bos. It includes diseases of cows, yaks, and zebus.Neoplasm Proteins: Proteins whose abnormal expression (gain or loss) are associated with the development, growth, or progression of NEOPLASMS. Some neoplasm proteins are tumor antigens (ANTIGENS, NEOPLASM), i.e. they induce an immune reaction to their tumor. Many neoplasm proteins have been characterized and are used as tumor markers (BIOMARKERS, TUMOR) when they are detectable in cells and body fluids as monitors for the presence or growth of tumors. Abnormal expression of ONCOGENE PROTEINS is involved in neoplastic transformation, whereas the loss of expression of TUMOR SUPPRESSOR PROTEINS is involved with the loss of growth control and progression of the neoplasm.Gene Expression Regulation, Neoplastic: Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control of gene action in neoplastic tissue.Gene Rearrangement, B-Lymphocyte, Heavy Chain: Ordered rearrangement of B-lymphocyte variable gene regions of the IMMUNOGLOBULIN HEAVY CHAINS, thereby contributing to antibody diversity. It occurs during the first stage of differentiation of the IMMATURE B-LYMPHOCYTES.Swine: Any of various animals that constitute the family Suidae and comprise stout-bodied, short-legged omnivorous mammals with thick skin, usually covered with coarse bristles, a rather long mobile snout, and small tail. Included are the genera Babyrousa, Phacochoerus (wart hogs), and Sus, the latter containing the domestic pig (see SUS SCROFA).DNA, Helminth: Deoxyribonucleic acid that makes up the genetic material of helminths.Herpesviridae Infections: Virus diseases caused by the HERPESVIRIDAE.Ligase Chain Reaction: A DNA amplification technique based upon the ligation of OLIGONUCLEOTIDE PROBES. The probes are designed to exactly match two adjacent sequences of a specific target DNA. The chain reaction is repeated in three steps in the presence of excess probe: (1) heat denaturation of double-stranded DNA, (2) annealing of probes to target DNA, and (3) joining of the probes by thermostable DNA ligase. After the reaction is repeated for 20-30 cycles the production of ligated probe is measured.Transfection: The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES.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.Neoplasm, Residual: Remnant of a tumor or cancer after primary, potentially curative therapy. (Dr. Daniel Masys, written communication)Gene Deletion: A genetic rearrangement through loss of segments of DNA or RNA, bringing sequences which are normally separated into close proximity. This deletion may be detected using cytogenetic techniques and can also be inferred from the phenotype, indicating a deletion at one specific locus.Cell Line, Tumor: A cell line derived from cultured tumor cells.Pedigree: The record of descent or ancestry, particularly of a particular condition or trait, indicating individual family members, their relationships, and their status with respect to the trait or condition.Macromolecular Substances: Compounds and molecular complexes that consist of very large numbers of atoms and are generally over 500 kDa in size. In biological systems macromolecular substances usually can be visualized using ELECTRON MICROSCOPY and are distinguished from ORGANELLES by the lack of a membrane structure.China: A country spanning from central Asia to the Pacific Ocean.Pregnancy: The status during which female mammals carry their developing young (EMBRYOS or FETUSES) in utero before birth, beginning from FERTILIZATION to BIRTH.RNA, Ribosomal, 16S: Constituent of 30S subunit prokaryotic ribosomes containing 1600 nucleotides and 21 proteins. 16S rRNA is involved in initiation of polypeptide synthesis.Chromosomes, Human, Pair 14: A specific pair of GROUP D CHROMOSOMES of the human chromosome classification.Cytomegalovirus: A genus of the family HERPESVIRIDAE, subfamily BETAHERPESVIRINAE, infecting the salivary glands, liver, spleen, lungs, eyes, and other organs, in which they produce characteristically enlarged cells with intranuclear inclusions. Infection with Cytomegalovirus is also seen as an opportunistic infection in AIDS.DNA, Ribosomal: DNA sequences encoding RIBOSOMAL RNA and the segments of DNA separating the individual ribosomal RNA genes, referred to as RIBOSOMAL SPACER DNA.Flow Cytometry: Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake.Genes, Immunoglobulin: Genes encoding the different subunits of the IMMUNOGLOBULINS, for example the IMMUNOGLOBULIN LIGHT CHAIN GENES and the IMMUNOGLOBULIN HEAVY CHAIN GENES. The heavy and light immunoglobulin genes are present as gene segments in the germline cells. The completed genes are created when the segments are shuffled and assembled (B-LYMPHOCYTE GENE REARRANGEMENT) during B-LYMPHOCYTE maturation. The gene segments of the human light and heavy chain germline genes are symbolized V (variable), J (joining) and C (constant). The heavy chain germline genes have an additional segment D (diversity).Heterozygote: An individual having different alleles at one or more loci regarding a specific character.Liver: A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances.Prevalence: The total number of cases of a given disease in a specified population at a designated time. It is differentiated from INCIDENCE, which refers to the number of new cases in the population at a given time.Myosin Heavy Chains: The larger subunits of MYOSINS. The heavy chains have a molecular weight of about 230 kDa and each heavy chain is usually associated with a dissimilar pair of MYOSIN LIGHT CHAINS. The heavy chains possess actin-binding and ATPase activity.Genes: A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms.Protozoan Infections, Animal: Infections with unicellular organisms formerly members of the subkingdom Protozoa. The infections may be experimental or veterinary.Introns: Sequences of DNA in the genes that are located between the EXONS. They are transcribed along with the exons but are removed from the primary gene transcript by RNA SPLICING to leave mature RNA. Some introns code for separate genes.Asian Continental Ancestry Group: Individuals whose ancestral origins are in the southeastern and eastern areas of the Asian continent.Swine Diseases: Diseases of domestic swine and of the wild boar of the genus Sus.Codon: A set of three nucleotides in a protein coding sequence that specifies individual amino acids or a termination signal (CODON, TERMINATOR). Most codons are universal, but some organisms do not produce the transfer RNAs (RNA, TRANSFER) complementary to all codons. These codons are referred to as unassigned codons (CODONS, NONSENSE).Immunoenzyme Techniques: Immunologic techniques based on the use of: (1) enzyme-antibody conjugates; (2) enzyme-antigen conjugates; (3) antienzyme antibody followed by its homologous enzyme; or (4) enzyme-antienzyme complexes. These are used histologically for visualizing or labeling tissue specimens.Chromosome Mapping: Any method used for determining the location of and relative distances between genes on a chromosome.Chromosomes, Human, Pair 18: A specific pair of GROUP E CHROMOSOMES of the human chromosome classification.Models, Molecular: Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.Horse Diseases: Diseases of domestic and wild horses of the species Equus caballus.Tumor Markers, Biological: Molecular products metabolized and secreted by neoplastic tissue and characterized biochemically in cells or body fluids. They are indicators of tumor stage and grade as well as useful for monitoring responses to treatment and predicting recurrence. Many chemical groups are represented including hormones, antigens, amino and nucleic acids, enzymes, polyamines, and specific cell membrane proteins and lipids.Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor: Ordered rearrangement of T-cell variable gene regions coding for the gamma-chain of antigen receptors.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.Molecular Weight: The sum of the weight of all the atoms in a molecule.Dogs: The domestic dog, Canis familiaris, comprising about 400 breeds, of the carnivore family CANIDAE. They are worldwide in distribution and live in association with people. (Walker's Mammals of the World, 5th ed, p1065)Carrier Proteins: Transport proteins that carry specific substances in the blood or across cell membranes.Disease Outbreaks: Sudden increase in the incidence of a disease. The concept includes EPIDEMICS and PANDEMICS.Herpesvirus 6, Human: The type species of ROSEOLOVIRUS isolated from patients with AIDS and other LYMPHOPROLIFERATIVE DISORDERS. It infects and replicates in fresh and established lines of hematopoietic cells and cells of neural origin. It also appears to alter NK cell activity. HHV-6; (HBLV) antibodies are elevated in patients with AIDS, Sjogren's syndrome, sarcoidosis, chronic fatigue syndrome, and certain malignancies. HHV-6 is the cause of EXANTHEMA SUBITUM and has been implicated in encephalitis.Tissue Fixation: The technique of using FIXATIVES in the preparation of cytologic, histologic, or pathologic specimens for the purpose of maintaining the existing form and structure of all the constituent elements.Parvoviridae Infections: Virus infections caused by the PARVOVIRIDAE.Leukocytes, Mononuclear: Mature LYMPHOCYTES and MONOCYTES transported by the blood to the body's extravascular space. They are morphologically distinguishable from mature granulocytic leukocytes by their large, non-lobed nuclei and lack of coarse, heavily stained cytoplasmic granules.Papillomavirus Infections: Neoplasms of the skin and mucous membranes caused by papillomaviruses. They are usually benign but some have a high risk for malignant progression.Herpesvirus 4, Human: The type species of LYMPHOCRYPTOVIRUS, subfamily GAMMAHERPESVIRINAE, infecting B-cells in humans. It is thought to be the causative agent of INFECTIOUS MONONUCLEOSIS and is strongly associated with oral hairy leukoplakia (LEUKOPLAKIA, HAIRY;), BURKITT LYMPHOMA; and other malignancies.Repetitive Sequences, Nucleic Acid: Sequences of DNA or RNA that occur in multiple copies. There are several types: INTERSPERSED REPETITIVE SEQUENCES are copies of transposable elements (DNA TRANSPOSABLE ELEMENTS or RETROELEMENTS) dispersed throughout the genome. TERMINAL REPEAT SEQUENCES flank both ends of another sequence, for example, the long terminal repeats (LTRs) on RETROVIRUSES. Variations may be direct repeats, those occurring in the same direction, or inverted repeats, those opposite to each other in direction. TANDEM REPEAT SEQUENCES are copies which lie adjacent to each other, direct or inverted (INVERTED REPEAT SEQUENCES).Fluorescent Antibody Technique, Direct: A form of fluorescent antibody technique utilizing a fluorochrome conjugated to an antibody, which is added directly to a tissue or cell suspension for the detection of a specific antigen. (Bennington, Saunders Dictionary & Encyclopedia of Laboratory Medicine and Technology, 1984)Rats, Sprague-Dawley: A strain of albino rat used widely for experimental purposes because of its calmness and ease of handling. It was developed by the Sprague-Dawley Animal Company.Infant, Newborn: An infant during the first month after birth.Cytomegalovirus Infections: Infection with CYTOMEGALOVIRUS, characterized by enlarged cells bearing intranuclear inclusions. Infection may be in almost any organ, but the salivary glands are the most common site in children, as are the lungs in adults.Polymorphism, Single Nucleotide: A single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population.Chlamydia Infections: Infections with bacteria of the genus CHLAMYDIA.Sequence Deletion: Deletion of sequences of nucleic acids from the genetic material of an individual.Disease Models, Animal: Naturally occurring or experimentally induced animal diseases with pathological processes sufficiently similar to those of human diseases. They are used as study models for human diseases.Clone Cells: A group of genetically identical cells all descended from a single common ancestral cell by mitosis in eukaryotes or by binary fission in prokaryotes. Clone cells also include populations of recombinant DNA molecules all carrying the same inserted sequence. (From King & Stansfield, Dictionary of Genetics, 4th ed)Membrane Proteins: Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.Homozygote: An individual in which both alleles at a given locus are identical.HeLa Cells: The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for VIRUS CULTIVATION and antitumor drug screening assays.Specimen Handling: Procedures for collecting, preserving, and transporting of specimens sufficiently stable to provide accurate and precise results suitable for clinical interpretation.Bone Marrow: The soft tissue filling the cavities of bones. Bone marrow exists in two types, yellow and red. Yellow marrow is found in the large cavities of large bones and consists mostly of fat cells and a few primitive blood cells. Red marrow is a hematopoietic tissue and is the site of production of erythrocytes and granular leukocytes. Bone marrow is made up of a framework of connective tissue containing branching fibers with the frame being filled with marrow cells.Aborted Fetus: A mammalian fetus expelled by INDUCED ABORTION or SPONTANEOUS ABORTION.RNA, Ribosomal, 18S: Constituent of the 40S subunit of eukaryotic ribosomes. 18S rRNA is involved in the initiation of polypeptide synthesis in eukaryotes.Herpesviridae: A family of enveloped, linear, double-stranded DNA viruses infecting a wide variety of animals. Subfamilies, based on biological characteristics, include: ALPHAHERPESVIRINAE; BETAHERPESVIRINAE; and GAMMAHERPESVIRINAE.DNA, Kinetoplast: DNA of kinetoplasts which are specialized MITOCHONDRIA of trypanosomes and related parasitic protozoa within the order KINETOPLASTIDA. Kinetoplast DNA consists of a complex network of numerous catenated rings of two classes; the first being a large number of small DNA duplex rings, called minicircles, approximately 2000 base pairs in length, and the second being several dozen much larger rings, called maxicircles, approximately 37 kb in length.Gene Expression Regulation, Enzymologic: Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control of gene action in enzyme synthesis.In Situ Hybridization, Fluorescence: A type of IN SITU HYBRIDIZATION in which target sequences are stained with fluorescent dye so their location and size can be determined using fluorescence microscopy. This staining is sufficiently distinct that the hybridization signal can be seen both in metaphase spreads and in interphase nuclei.Prognosis: A prediction of the probable outcome of a disease based on a individual's condition and the usual course of the disease as seen in similar situations.Saccharomyces cerevisiae: A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement.BrazilFluorescent Antibody Technique, Indirect: A form of fluorescent antibody technique commonly used to detect serum antibodies and immune complexes in tissues and microorganisms in specimens from patients with infectious diseases. The technique involves formation of an antigen-antibody complex which is labeled with fluorescein-conjugated anti-immunoglobulin antibody. (From Bennington, Saunders Dictionary & Encyclopedia of Laboratory Medicine and Technology, 1984)Predictive Value of Tests: In screening and diagnostic tests, the probability that a person with a positive test is a true positive (i.e., has the disease), is referred to as the predictive value of a positive test; whereas, the predictive value of a negative test is the probability that the person with a negative test does not have the disease. Predictive value is related to the sensitivity and specificity of the test.Down-Regulation: A negative regulatory effect on physiological processes at the molecular, cellular, or systemic level. At the molecular level, the major regulatory sites include membrane receptors, genes (GENE EXPRESSION REGULATION), mRNAs (RNA, MESSENGER), and proteins.Prospective Studies: Observation of a population for a sufficient number of persons over a sufficient number of years to generate incidence or mortality rates subsequent to the selection of the study group.Genome, Viral: The complete genetic complement contained in a DNA or RNA molecule in a virus.Bacteriophage T7: Virulent bacteriophage and type species of the genus T7-like phages, in the family PODOVIRIDAE, that infects E. coli. It consists of linear double-stranded DNA, terminally redundant, and non-permuted.Chlamydia trachomatis: Type species of CHLAMYDIA causing a variety of ocular and urogenital diseases.DNA Methylation: Addition of methyl groups to DNA. DNA methyltransferases (DNA methylases) perform this reaction using S-ADENOSYLMETHIONINE as the methyl group donor.Mycoplasma Infections: Infections with species of the genus MYCOPLASMA.Cat Diseases: Diseases of the domestic cat (Felis catus or F. domesticus). This term does not include diseases of the so-called big cats such as CHEETAHS; LIONS; tigers, cougars, panthers, leopards, and other Felidae for which the heading CARNIVORA is used.Bacteriological Techniques: Techniques used in studying bacteria.Digoxigenin: 3 beta,12 beta,14-Trihydroxy-5 beta-card-20(22)-enolide. A cardenolide which is the aglycon of digoxin. Can be obtained by hydrolysis of digoxin or from Digitalis orientalis L. and Digitalis lanata Ehrh.Genes, ras: Family of retrovirus-associated DNA sequences (ras) originally isolated from Harvey (H-ras, Ha-ras, rasH) and Kirsten (K-ras, Ki-ras, rasK) murine sarcoma viruses. Ras genes are widely conserved among animal species and sequences corresponding to both H-ras and K-ras genes have been detected in human, avian, murine, and non-vertebrate genomes. The closely related N-ras gene has been detected in human neuroblastoma and sarcoma cell lines. All genes of the family have a similar exon-intron structure and each encodes a p21 protein.Temperature: The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.Mice, Inbred C57BLFatal Outcome: Death resulting from the presence of a disease in an individual, as shown by a single case report or a limited number of patients. This should be differentiated from DEATH, the physiological cessation of life and from MORTALITY, an epidemiological or statistical concept.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).

*  Polymerase chain reaction-based detection of Mycobacterium tuberculosis DNA in formalin-fixed, paraffin-embedded skin tissue...

Skin and Cancer Foundation, Inc., Pasig City, Philippines, and the National Institute of Molecular Biology and Biotechnology, College of Science, University of the Philippines, ...
onlinelibrary.wiley.com/doi/10.1111/j.1365-4632.2010.04189.x/full

*  A semi-quantitative reverse transcriptase polymerase chain reaction method for measurement of mRNA for TNF-alpha and IL-1beta...

A semi-quantitative reverse transcriptase polymerase chain reaction method for measurement of mRNA for TNF-alpha and IL-1beta in whole blood cultures: its application in typhoid fever and exentric ...
repository.ubn.ru.nl/handle/2066/23983

*  A power-efficient thermocycler based on induction heating for DNA amplification by polymerase chain reaction - ePrints@IISc

We have built a thermocycler based on the principles of induction heating for polymerase chain reaction (PCR) of target sequences in DNA samples of interest. The cycler has an average heating rate of ~0.8 �°C/s and a cooling rate of ~0.5 �°C/s, and typically takes ~4 h to complete a 40-cycle PCR protocol. It is power-efficient (~6 W per reaction tube), micro-processor controlled, and can be adapted for battery operation. Using this instrument, we have successfully amplified a 350 bp segment from a plasmid and SRY, the human sex determining gene, which occurs as a single-copy sequence in genomic DNA of human males. The PCR products from this thermocycler are comparable to those obtained by the use of commercially available machines. Its easy front-end operation, low-power design, portability and low cost makes it suitable for diagnostic field applications of PCR.. ...
eprints.iisc.ernet.in/2753/

*  PCR Links.com -The Web Guide Of Polymerase Chain Reaction Technique - PCR

Polymerase Chain Reaction (PCR):Selected links about PCR methods: Asymmetric, Colony, DD, Degenerate, Hot Start, Inverse, In situ, Long, Multiplex, Nested, QC, RACE, RAPD, Real-Time, Rep, RFLP, RT, SSCP, Touchdown and more.
pcrlinks.com

*  HID Veriti 96-Well Thermal Cycler, 0.2 mL - Thermo Fisher Scientific

Building upon the proven reliability and performance of the Applied Biosystems 9600 and 9700 GeneAmp Thermal Cycler instruments, the HID Veriti Thermal Cycler features a 96-well (0.2 mL) format with six independent temperature zones to provide precise temperature control and rapid temperature change
thermofisher.com/order/catalog/product/4479071

*  Conventional Thermal Cyclers<i class='icon'...

Esco offers a choice of Conventional Thermal Cycler and Real Time Thermal Cycler models designed to meet critical requirements for all kinds of PCR processes
escoglobal.com.ph/category/thermal-cyclers/

*  Esco | MiniPro Thermal Cyclers

Discover the fastest personal thermal cyclers in the world market today, the Swift™ MiniPro Thermal Cyclers which has outstanding ramp rates of up to 5 °C.
escoglobal.com.ph/product/real-time-pcr-systems/minipro-thermal-cyclers/swt-mip/

*  Automation of Polymerase Chain Reaction-Based Systems for Detection of Hepatitis C Virus RNA : Clinical Chemistry and...

Polymerase chain reaction-based molecular assays are gaining increasing importance in the diagnosis and monitoring of infectious diseases. Over the past several years, the development and application of these techniques has initiated a revolution in the diagnosis and monitoring of hepatitis C infection. Presently, molecular assays are exclusively done in especially dedicated laboratories. Advances in automation will bring these technologies into routine diagnostic laboratories and will make them widely used. ...
https://degruyter.com/view/j/cclm.1998.36.issue-8/cclm.1998.100/cclm.1998.100.xml

*  Molecular Pathology | Clinical Gate

There is a long and growing list of gene translocation events that are linked to cancer. Whether the result of intra- or interchromosomal exchanges, these translocations commonly involve genes encoding a kinase or a transcription factor. The resulting fusion genes are often the principal drivers of tumorigenesis and therefore serve as diagnostic markers and/or targets for specific therapies (e.g., kinase inhibitors). Fusion mRNAs from translocation events can be detected by highly sensitive methodologies based on polymerase chain reaction (PCR); however, these approaches can be frustrated by the fact that a particular target gene may be fused to any of more than a dozen different partner genes, requiring numerous primers to cover all possible fusion events. In contrast, FISH can detect a translocation-related break in a target gene irrespective of which partner gene has been fused to it. This is done by labeling two pools of probes with different ...
https://clinicalgate.com/molecular-pathology-2/

*  RT² qPCR Primer Assay for Rat Mapk8

The RT² qPCR Primer Assay is the most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis. Our experimentally verified design algorithm yields gene-specific qPCR assays characterized by uniform and high PCR efficiencies and standardized amplification conditions. Every RT² Primer Assay is subjected to rigorous experimental verification. Single product amplification of the correct size and high PCR efficiency are guaranteed when using the appropriate RT² qPCR Master Mixes. The uniform PCR amplification efficiencies and PCR conditions of the RT² qPCR Primer Assays provide an accurate and scalable solution for multiple gene expression analyses. Browse Primer Assays By Gene ...
sabiosciences.com/primerinfo.php?pcatn=PPR43333A

*  RT² qPCR Primer Assay for Human FGB

The RT² qPCR Primer Assay is the most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis. Our experimentally verified design algorithm yields gene-specific qPCR assays characterized by uniform and high PCR efficiencies and standardized amplification conditions. Every RT² Primer Assay is subjected to rigorous experimental verification. Single product amplification of the correct size and high PCR efficiency are guaranteed when using the appropriate RT² qPCR Master Mixes. The uniform PCR amplification efficiencies and PCR conditions of the RT² qPCR Primer Assays provide an accurate and scalable solution for multiple gene expression analyses. Browse Primer Assays By Gene ...
sabiosciences.com/primerinfo.php?pcatn=PPH00814A

*  RT² qPCR Primer Assay for Mouse Dsg4

The RT² qPCR Primer Assay is the most reliable SYBR® Green-based quantitative real-time PCR assay for gene expression analysis. Our experimentally verified design algorithm yields gene-specific qPCR assays characterized by uniform and high PCR efficiencies and standardized amplification conditions. Every RT² Primer Assay is subjected to rigorous experimental verification. Single product amplification of the correct size and high PCR efficiency are guaranteed when using the appropriate RT² qPCR Master Mixes. The uniform PCR amplification efficiencies and PCR conditions of the RT² qPCR Primer Assays provide an accurate and scalable solution for multiple gene expression analyses. Browse Primer Assays By Gene ...
sabiosciences.com/primerinfo.php?pcatn=PPM36209A

*  Polymerase Chain Reaction - PCR Technologies Guide | Sigma-Aldrich

The polymerase chain reaction (PCR) has become one of the most widely used techniques in molecular biology. It is used in applications from basic research to high-throughput screening.
sigmaaldrich.com/technical-documents/articles/biology/polymerase-chain-reaction.html

*  TOPO TA CLONING - LIGATION STEP - Molecular Cloning

First question - have you had success with TOPO TA cloning in the past or is this your first time?. If it is your first time using TOPO TA then there could be many different causes for your problem. If you haven't already, I suggest you try the control cloning. Not the plasmid control (only controls for transformation) but the control that allows you to create a PCR product, clone and transform. Then you can determine if the problem is technique related. However, if you have been successful then start by reviewing the troubleshooting guide in the back of Invitrogen's product insert. Problem could be due to:. 1. Bad primer design = redesign primers. 2. PCR Contaminant or non specific PCR product = gel purify. 3. Cloned PCR product is toxic to bacteria = if possible amplify a different region or try different competent cells to transform. 4. PCR product is poor quality = purify and reduce UV exposure, and use fresh PCR product. 5. Too much PCR product used - try 3:1 insert to vector ratio. 6. ...
protocol-online.org/biology-forums-2/posts/28236.html

*  problem of restriction enzyme digestion - Molecular Cloning - BioForum

problem of restriction enzyme digestion - posted in Molecular Cloning: hi all, i am doing SNP genotyping. my pcr product is 280 bp if the subject is homozygous wildtype, the RE will not cut, so i will get a single 280 bp band if the subject is homozygous variant, the RE will cut, and i will get a 240 bp band and a 40 bp band if the subject is heterozygote, i will get all 280 bp, 240 bp and 40 bp bands now i have one problem i cannot be sure whether the RE cuts or not for some samples, t...
protocol-online.org/forums/topic/25896-problem-of-restriction-enzyme-digestion/

*  Lysis buffer selection - SDS-PAGE and Western Blotting

All of the above really. For tissue you will need a fairly strong lysis buffer to get the proteins out compared to what you would need to lyse cultured cells. Some proteins are not readily soluble so you need to add more detergents to get them to solubilise. Some reagents, usually detergents interfere with protein quantitation with particular assays. Some antibodies detect only native protein, some only denatured protein...the lysis buffer can affect the conformation of the protein ...
protocol-online.org/biology-forums-2/posts/17626.html

*  Bio-Resource: Helicase Dependent Amplification...DNA Amplification Without Thermocycler

Helicase Dependent Amplification (HDA) is an invitro nucleic acid amplificatio method similar to Polymerase Chain Reaction (PCR).
technologyinscience.blogspot.com/2013/07/helicase-dependent-amplificationdna.html

*  C3orf67 - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's C3orf67 primer pair, for Human. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dhsaeg5016845-primepcr-ddpcr-expression-evagreen-assay-c3orf67-human

*  LAMP3 - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's LAMP3 primer pair, for Human. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dhsaeg5007344-primepcr-ddpcr-expression-evagreen-assay-lamp3-human

*  MPV17L - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's MPV17L primer pair, for Human. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dhsaeg5016624-primepcr-ddpcr-expression-evagreen-assay-mpv17l-human

*  CDK5RAP3 - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's CDK5RAP3 primer pair, for Human. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dhsaeg5017700-primepcr-ddpcr-expression-evagreen-assay-cdk5rap3-human

*  HOXB8 - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's HOXB8 primer pair, for Human. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dhsaeg5000568-primepcr-ddpcr-expression-evagreen-assay-hoxb8-human

*  SLC25A32 - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's SLC25A32 primer pair, for Human. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dhsaeg5010716-primepcr-ddpcr-expression-evagreen-assay-slc25a32-human

*  Ces2c - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's Ces2c primer pair, for Mouse. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dmmueg5074920-primepcr-ddpcr-expression-evagreen-assay-ces2c-mouse

*  FKBP8 - ddPCR Primer Pair - EvaGreen - Digital PCR | PrimePCR | Bio-Rad

Perform reliable PCR with Bio-Rad's FKBP8 primer pair, for Human. Designed for EvaGreen-based detection with digital PCR (ddPCR).
bio-rad.com/en-us/prime-pcr-assays/assay/dhsaeg5014457-primepcr-ddpcr-expression-evagreen-assay-fkbp8-human

Thermal cyclerSymmetry element: A symmetry element is a point of reference about which symmetry operations can take place. In particular, symmetry elements can be centers of inversion, axes of rotation and mirror planes.Coles PhillipsT7 DNA polymerase: T7 DNA polymerase is an enzyme used during the DNA replication of the T7 bacteriophage. During this process, the DNA polymerase “reads” existing DNA strands and creates two new strands that match the existing ones.Mature messenger RNA: Mature messenger RNA, often abbreviated as mature mRNA is a eukaryotic RNA transcript that has been spliced and processed and is ready for translation in the course of protein synthesis. Unlike the eukaryotic RNA immediately after transcription known as precursor messenger RNA, it consists exclusively of exons, with all introns removed.Transcription preinitiation complex: The preinitiation complex (abbreviated PIC) is a large complex of proteins that is necessary for the transcription of protein-coding genes in eukaryotes (+archaea). The preinitiation complex helps position RNA polymerase II over gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site for transcription.Assay sensitivity: Assay sensitivity is a property of a clinical trial defined as the ability of a trial to distinguish an effective treatment from a less effective or ineffective intervention. Without assay sensitivity, a trial is not internally valid and is not capable of comparing the efficacy of two interventions.Protein primary structure: The primary structure of a peptide or protein is the linear sequence of its amino acid structural units, and partly comprises its overall biomolecular structure. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end.DNA condensation: DNA condensation refers to the process of compacting DNA molecules in vitro or in vivo. Mechanistic details of DNA packing are essential for its functioning in the process of gene regulation in living systems.Ligation-independent cloning: Ligation-independent cloning (LIC) is a form of molecular cloning that is able to be performed without the use of restriction endonucleases or DNA ligase. This allows genes that have restriction sites to be cloned without worry of chopping up the insert.Core enzyme: A core enzyme consists of the subunits of an enzyme that are needed for catalytic activity, as in the core enzyme RNA polymerase.Genetics: Analysis & Principles, 3rd Edition.Eukaryotic transcription: Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells.Klenow fragmentAllele-specific oligonucleotide: An allele-specific oligonucleotide (ASO) is a short piece of synthetic DNA complementary to the sequence of a variable target DNA. It acts as a probe for the presence of the target in a Southern blot assay or, more commonly, in the simpler Dot blot assay.Silent mutation: Silent mutations are mutations in DNA that do not significantly alter the phenotype of the organism in which they occur. Silent mutations can occur in non-coding regions (outside of genes or within introns), or they may occur within exons.Multiplex polymerase chain reaction: 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.Amplified fragment length polymorphismHolE: In E. coli and other bacteria, holE is a gene that encodes the theta subunit of DNA polymerase III.DNA sequencer: A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample of DNA, a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A (adenine) and T (thymine).Regenerative amplification: In laser science, regenerative amplification is a process used to generate short but strong pulses of laser light. It is based on a pulse trapped in a laser resonator, which stays in there until it extracts all of the energy stored in the amplification medium.Gene polymorphismInfinite alleles model: The infinite alleles model is a mathematical model for calculating genetic mutations. The Japanese geneticist Motoo Kimura and American geneticist James F.Immunoglobulin heavy chainList of strains of Escherichia coli: Escherichia coli is a well studied bacterium that was first identified by Theodor Escherich, after whom it was later named.GC box: In molecular biology, a GC box is a distinct pattern of nucleotides found in the promoter region of some eukaryotic genes upstream of the TATA box and approximately 110 bases upstream from the transcription initiation site. It has a consensus sequence GGGCGG which is position dependent and orientation independent.Alternative splicing: Alternative splicing is a regulated process during gene expression that results in a single gene coding for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene.Branching order of bacterial phyla (Gupta, 2001): There are several models of the Branching order of bacterial phyla, one of these was proposed in 2001 by Gupta based on conserved indels or protein, termed "protein signatures", an alternative approach to molecular phylogeny. Some problematic exceptions and conflicts are present to these conserved indels, however, they are in agreement with several groupings of classes and phyla.YjdF RNA motifCS-BLASTBeef cattle: Beef cattle are cattle raised for meat production (as distinguished from dairy cattle, used for milk production). The meat of adult cattle is known as beef.Gene signature: A gene signature is a group of genes in a cell whose combined expression patternItadani H, Mizuarai S, Kotani H. Can systems biology understand pathway activation?Nested case-control study: A nested case control (NCC) study is a variation of a case-control study in which only a subset of controls from the cohort are compared to the incident cases. In a case-cohort study, all incident cases in the cohort are compared to a random subset of participants who do not develop the disease of interest.Temporal analysis of products: Temporal Analysis of Products (TAP), (TAP-2), (TAP-3) is an experimental technique for studyingPoint mutationCpG OligodeoxynucleotideChromothripsis: Chromothripsis is the phenomenon by which up to thousands of clustered chromosomal rearrangements occur in a single event in localised and confined genomic regions in one or a few chromosomes, and is known to be involved in both cancer and congenital diseases. It occurs through one massive genomic rearrangement during a single catastrophic event in the cell's history.Burst kinetics: Burst kinetics is a form of enzyme kinetics that refers to an initial high velocity of enzymatic turnover when adding enzyme to substrate. This initial period of high velocity product formation is referred to as the "Burst Phase".Immunoglobulin light chainSingle-strand conformation polymorphism: Single-strand conformation polymorphism (SSCP), or single-strand chain polymorphism, is defined as conformational difference of single-stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions. This property allows sequences to be distinguished by means of gel electrophoresis, which separates fragments according to their different conformations.Polymerase-endonuclease amplification reaction: Polymerase-endonuclease amplification reaction (PEAR) is a DNA amplification technology for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides.Eva Engvall: Eva Engvall, born 1940, is one of the scientists who invented ELISA in 1971.Eva Engvall, The Scientist 1995, 9(18):8Pituitary-specific positive transcription factor 1: POU domain, class 1, transcription factor 1 (Pit1, growth hormone factor 1), also known as POU1F1, is a transcription factor for growth hormone.Phenotype microarray: The phenotype microarray approach is a technology for high-throughput phenotyping of cells.PolymeraseDNA re-replication: DNA re-replication (or simply rereplication) is an undesirable and possibly fatal occurrence in eukaryotic cells in which the genome is replicated more than once per cell cycle. Rereplication is believed to lead to genomic instability and has been implicated in the pathologies of a variety of human cancers.Oncogene: An oncogene is a gene that has the potential to cause cancer.Wilbur, Beth, editor.PSI-6130Old German Shepherd Dog: Old German Shepherd Dog () is a controversial predicate for the long-hair variation of the German Shepherd Dog (), which is not a separate breed recognized by the Fédération Cynologique Internationale. Nonetheless, there are efforts to establish this variety as a separate breed.Genetic variation: right|thumbGeneralizability theory: Generalizability theory, or G Theory, is a statistical framework for conceptualizing, investigating, and designing reliable observations. It is used to determine the reliability (i.Cellular microarray: A cellular microarray is a laboratory tool that allows for the multiplex interrogation of living cells on the surface of a solid support. The support, sometimes called a "chip", is spotted with varying materials, such as antibodies, proteins, or lipids, which can interact with the cells, leading to their capture on specific spots.DNA-binding proteinBrain biopsyMargaret Jope: Margaret Jope (1913–2004) was a Scottish biochemist, born as Henrietta Margaret Halliday in Peterhead, Scotland.DNA binding site: DNA binding sites are a type of binding site found in DNA where other molecules may bind. DNA binding sites are distinct from other binding sites in that (1) they are part of a DNA sequence (e.Ferric uptake regulator family: In molecular biology, the ferric uptake regulator (FUR) family of proteins includes metal ion uptake regulator proteins. These are responsible for controlling the intracellular concentration of iron in many bacteria.

(1/67158) Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer.

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.  (+info)

(2/67158) Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells.

DNA-dependent protein kinase (DNA-PK) functions in double-strand break repair and immunoglobulin [V(D)J] recombination. We previously established a radiation-sensitive human cell line, M059J, derived from a malignant glioma, which lacks the catalytic subunit (DNA-PKcs) of the DNA-PK multiprotein complex. Although previous Northern blot analysis failed to detect the DNA-PKcs transcript in these cells, we show here through quantitative studies that the transcript is present, albeit at greatly reduced (approximately 20x) levels. Sequencing revealed no genetic alteration in either the promoter region, the kinase domain, or the 3' untranslated region of the DNA-PKcs gene to account for the reduced transcript levels. Nuclear run-on transcription assays indicated that the rate of DNA-PKcs transcription in M059J and DNA-PKcs proficient cell lines was similar, but the stability of the DNA-PKcs message in the M059J cell line was drastically (approximately 20x) reduced. Furthermore, M059J cells lack an alternately spliced DNA-PKcs transcript that accounts for a minor (5-20%) proportion of the DNA-PKcs message in all other cell lines tested. Thus, alterations in DNA-PKcs mRNA stability and/or the lack of the alternate mRNA may result in the loss of DNA-PKcs activity. This finding has important implications as DNA-PKcs activity is essential to cells repairing damage induced by radiation or radiomimetric agents.  (+info)

(3/67158) Hybrid capture II, a new sensitive test for human papillomavirus detection. Comparison with hybrid capture I and PCR results in cervical lesions.

AIM: To test a new assay for the detection of human papillomavirus (HPV) DNA, hybrid capture II (HC II), compared with the previous commercialized hybrid capture I (HC I) and polymerase chain reaction (PCR) results on cervical scrapes from fresh cone excision biopsy samples. METHODS: The three methods were used on cervical scrapes from 42 fresh cone excision biopsy samples. There were nine metaplastic and inflammatory lesions, five low grade lesions, and 28 high grade lesions. PCR was performed using the general primers GP5+/GP6+. The viral load of high risk HPV DNA was estimated by the ratio of relative light units to positive control values in the samples. RESULTS: The sensitivity of HC I for the detection of high grade lesions was 71.4%, while it was 92.8% for HC II and 96.4% for the PCR. Considering only the absence of detectable cervical in situ neoplasia, the specificity was 88.9% for HC I, 66.7% for HC II, and 66.7% for PCR. With HC II, for a ratio of cervical sample to normal control of > 200, the sensitivity for the detection of high grade lesion was only 34.6% with a specificity of 66.7%. CONCLUSIONS: HPV detection with the HC II assay is more sensitive than the previous HC I and represents a more convenient and easier test than PCR for routine use. Nevertheless the viral load estimated with this test cannot be a reliable predictive indicator of high grade lesions.  (+info)

(4/67158) Human papillomavirus DNA in adenosquamous carcinoma of the lung.

AIM: To investigate the presence of human papillomavirus (HPV) DNA in adenosquamous carcinoma of the lung--which is relatively common in Okinawa but not in mainland Japan--and examine its histological features. METHODS: Of 207 cases where primary lung cancers were surgically removed between January 1995 and June 1997 in Okinawa, 23 were adenosquamous carcinoma. HPV was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for E6 and E7 regions of the HPV genome. PCR products were analysed by Southern blotting. Immunohistochemical determination of high molecular weight cytokeratin (HMC) and involucrin was also carried out. RESULTS: 18 cases were positive for HPV DNA by PCR and NISH. HPV types 6, 11, 16, and 18 were found. Seven cases were dual positive for different types of HPV. Using NISH, HPV was also found in the squamous cell components and in neighbouring enlarged adenocarcinoma cells. The HMC and involucrin were demonstrated immunohistochemically in the same areas. CONCLUSIONS: HPV DNA was found in a high proportion (78.3%) of adenosquamous carcinomas in Okinawa, a region where HPV has previously been shown to be prevalent in squamous cell carcinoma of the lung. The adenocarcinoma cells adjacent to the squamous cell carcinoma component were enlarged and positive for HPV, HMC, and involucrin. This is thought to indicate the transition from adenocarcinoma to squamous cell carcinoma.  (+info)

(5/67158) Immunohistochemical expression of mdm2 and p21WAF1 in invasive cervical cancer: correlation with p53 protein and high risk HPV infection.

AIM: To investigate the immunocytochemical staining pattern of mdm2 and p21WAF1 proteins in invasive cervical cancer and to determine its relation with the expression of p53 and with the high risk HPV infection. METHODS: Immunocytochemistry for p53, mdm2, and p21WAF1 was performed in 31 paraffin embedded sections of invasive cervical cancer. The results were assessed by image analysis, evaluating for each protein the optical density of the immunostained area, scored as percentage of the total nuclear area. The presence of high risk human papillomavirus (HPV) infection was detected by using the polymerase chain reaction. RESULTS: Immunostaining for both mdm2 and p21WAF1 was correlated with p53 expression; however, the correlation between p53 and mdm2 (R = 0.49; p < 0.01) was more significant than between p53 and p21WAF1 (R = 0.31; p < 0.05); the less stringent correlation between p53 and p21WAF1 might reflect the p53 independent mechanisms of p21WAF1 induction. Similar average levels of p53, mdm2, and p21WAF1 immunostaining were found in the presence or absence of high risk HPV-DNA, without significant differences between the two groups. CONCLUSIONS: These data suggest that mdm2 and p21WAF1 proteins are expressed in invasive cervical cancer and that their immunocytochemical staining pattern is not abrogated by the presence of high risk HPV genomic sequences.  (+info)

(6/67158) The significance of cagA and vacA subtypes of Helicobacter pylori in the pathogenesis of inflammation and peptic ulceration.

AIMS: To assess the significance of cagA and vacA subtypes of Helicobacter pylori in relation to inflammation and density of bacterial colonisation in vivo within a dyspeptic UK population. METHODS: Dyspeptic patients who were Helicobacter pylori positive had antral samples taken for histology and culture. Gastroduodenal pathology was noted. The grade of bacterial density and inflammation was assessed using the Sydney system. Bacterial DNA was extracted and the vacA alleles and the cagA/gene typed using PCR. RESULTS: 120 patients were studied. There was high rate of cagA positive strains in this population. Bacterial density did not correlate with the presence of peptic ulceration. There was a significant association between cagA positive strains and increased inflammation and bacterial density. The vacA s1 type independently correlated with extensive chronic inflammation but there was no association with bacterial density. The vacA m type did not correlate with extent of inflammation or bacterial density. CONCLUSIONS: The results suggest that cagA is important in the pathogenesis of inflammation and peptic ulceration. These findings are in keeping with the hypothesis that cagA acts as a marker for a cag pathogenicity island which encodes several genes involved in inflammation. The vacA s1 allele correlates with inflammation independently of cagA, possibly through its enhanced ability to produce the vacuolating cytotoxin.  (+info)

(7/67158) Expression of vascular endothelial growth factor in human oral squamous cell carcinoma: its association with tumour progression and p53 gene status.

AIMS: To correlate vascular endothelial growth factor (VEGF) expression in oral squamous cell carcinoma with the clinicopathological characteristics and prognosis; and to assess whether p53 gene status is associated with VEGF expression in human cancers. METHODS: Tumour specimens from 45 patients with oral squamous cell carcinomas were examined. Expression of VEGF was determined using an immunohistochemical method, and a tumour was considered positive when more than 5% of the neoplastic cells showed VEGF immunoreactivity. The p53 gene status was screened using a polymerase chain reaction--single strand conformation polymorphism analysis. RESULTS: VEGF positive staining was detected in 19 (42.2%) of the 45 cases. VEGF immunoreactivity did not correlate with the histological degree of tumour differentiation, clinical stages, or lymph node metastasis. The patients with VEGF positive tumours had a significantly worse prognosis than those with VEGF negative tumours. The five year overall survival rate of the VEGF negative patients was 76.5%, as compared with 48.8% for the VEGF positive patients. No significant association between VEGF expression and the p53 gene status of the tumours was found. CONCLUSIONS: VEGF is a good prognostic indicator of the survival of patients with oral squamous cell carcinoma. The p53 gene status does not seem to be associated with VEGF expression in these cancers.  (+info)

(8/67158) The alphaE-catenin gene (CTNNA1) acts as an invasion-suppressor gene in human colon cancer cells.

The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the alphaE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second alphaE-catenin allele. The alphaE-catenin gene is therefore, an invasion-suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes.  (+info)



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