|
Variants Of PCR
The versatility of polymerase chain reaction (PCR) has led to a large number of variants of PCR. Basic modifications Often only a small modification needs to be made to the standard PCR protocol to achieve a desired goal: ''Multiplex-PCR'' uses several pairs of primers annealing to different target sequences. This permits the simultaneous analysis of multiple targets in a single sample. For example, in testing for genetic mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the targets assayed are often amplified in groups of 3 or 4. ''Multiplex Ligation-dependent Probe Amplification'' (or ''Multiplex Ligation-dependent Probe Amplification, MLPA'') permits multiple targets to be amplified using only a single pair of primers, avoiding the resolution limitations of multiplex PCR. Multiplex PCR has also been used for analysis of Microsatellite (genetics), microsatellites and single nucleotide polymorphism, SNPs. ''Variable N ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith (chemist), Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993. PCR is fundamental to many of the procedures used in genetic testing and research, including analysis of Ancient DNA, ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Overlap-extension PCR
The overlap extension polymerase chain reaction (or OE-PCR) is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension (SOE) PCR. It is used to insert specific mutations at specific points in a sequence or to splice smaller DNA fragments into a larger polynucleotide. Splicing of DNA Molecules As in most PCR reactions, two primers—one for each end—are used per sequence. To splice two DNA molecules, special primers are used at the ends that are to be joined. For each molecule, the primer at the end to be joined is constructed such that it has a 5' overhang complementary to the end of the other molecule. Following annealing when replication occurs, the DNA is extended by a new sequence that is complementary to the molecule it is to be joined to. Once both DNA molecules are extended in such a manner, they are mixed and a PCR is carried out with only the primers for the far ends. The overlapping complementary sequences introduced ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Polony (biology)
Polony is a contraction of "polymerase colony," a small colony of DNA. Polonies are discrete clonal amplifications of a single DNA molecule, grown in a gel matrix. This approach greatly improves the signal-to-noise ratio. Polonies can be generated using several techniques that include solid-phase polymerase chain reaction (PCR]) in polyacrylamide gels. However, other earlier patented technologies, such as that from Manteia Predictive Medicine (acquired by Solexa), which generate DNA on a solid phase surface by bridge amplification - are generally referred to as "clusters". The terminology and distinction between 'polony' and 'cluster' have become confused recently. Growth of clonal copies of DNA on bead surfaces remains to be generically named although some also seek to name this technique as a "polony" method. The concept of localizing and analyzing regions containing clonal nucleic acid populations was first described in patents by Brown, et al.. (assigned to Genomic Nanosystem ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Digital Polymerase Chain Reaction
Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. A "digital" measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the inter ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
DNA Ligase
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as DNA ligase IV) may specifically repair double-strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA. DNA ligase is used in both DNA repair and DNA replication (see '' Mammalian ligases''). In addition, DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments (see '' Research applications''). Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA. Enzymatic mechanism The mechanism of DNA ligase is to form two covalent phos ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Polymerase Cycling Assembly
Polymerase cycling assembly (or PCA, also known as Assembly PCR) is a method for the assembly of large DNA oligonucleotides from shorter fragments. The process uses the same technology as PCR, but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process. It thus allows for the production of synthetic genes and even entire synthetic genomes. PCA principles Much like how primers are designed such that there is a forward primer and a reverse primer capable of allowing DNA polymerase to fill the entire template sequence, PCA uses the same technology but with multiple oligonucleotides. While in PCR the customary size of oligonucleotides used is 18 base pairs, in PCA lengths of up to 50 are used to ensure uniqueness and correct hybridization. Each oligonucleotide is designed to be either part of the top or bottom strand of the target sequenc ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Touchdown Polymerase Chain Reaction
The touchdown polymerase chain reaction or touchdown style polymerase chain reaction is a method of polymerase chain reaction by which primers avoid amplifying nonspecific sequences. The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just above this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will "swamp out" any specific sequences because of the exponential Exponential may refer to any of several mathematical topics related to exponentiation, including: *Exponential function, also: **Matrix exponential, the matrix analogue to the above *Exponential decay, decrease at a rate pr ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Antibody
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can ''tag'' a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly (for example, by blocking a part of a virus that is essential for its invasion). To allow the immune system to recognize millions of different antigens, the antigen-binding sites at both tips of the antibody come in an equally wide variety. In contrast, the remainder of the antibody is relatively constant. It only occurs in a few varia ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
TaqMan
TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-La Roche for diagnostic assays and by Applied Biosystems (now part of Thermo Fisher Scientific) for research applications. The TaqMan probe principle relies on the 5´–3´ exonuclease activity of ''Taq'' polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection. As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection. TaqMan probes were named after the videogame Pac-Man (''Taq'' Polymerase + PacMan = TaqMan) as its mechanism is based on the Pac-Man principle ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Fluorophore
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds. Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environmental aspects such as polarity or ions). More generally they are covalently bonded to a macromolecule, serving as a marker (or dye, or tag, or reporter) for affine or bioactive reagents (antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, i.e., fluorescent imaging and spectroscopy. Fluorescein, via its amine-reactive isothiocyanate derivative fluorescein isothiocyanate (FITC), has been one of the most popular fluorophores. Fro ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR. Real-time PCR can be used quantitatively (quantitative real-time PCR) and semi-quantitatively (i.e., above/below a certain amount of DNA molecules) (semi-quantitative real-time PCR). Two common methods for the detection of PCR products in real-time PCR are (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments ( MIQE) guidelines propose that the abbreviation ''qPCR'' be used for q ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
.svg "Click for more on -> Plasmid")
