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A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) 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.
The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. [2] [3] The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP.
The free NCBI tool Primer-BLAST integrates primer design and BLAST search into one application, [6] as do commercial software products such as ePrime and Beacon Designer. Computer simulations of theoretical PCR results ( Electronic PCR ) may be performed to assist in primer design by giving melting and annealing temperatures, etc. [ 7 ]
Tailed-primers include non-complementary sequences at their 5' ends. A common procedure is the use of linker-primers, which ultimately place restriction sites at the ends of the PCR products, facilitating their later insertion into cloning vectors. An extension of the 'colony-PCR' method (above), is the use of vector primers.
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 nature of polymerase amplification.
Loop-mediated isothermal amplification (LAMP) primers [1] Loop-mediated isothermal amplification (LAMP) product [1]. In LAMP, the target sequence is amplified at a constant temperature of 60–65 °C (140–149 °F) using either two or three sets of primers and a polymerase like Bst Klenow fragment with high strand displacement activity in addition to a replication activity.
The GFP-technique involves altering the genetic information of the cells. [ 18 ] [ 19 ] A significant problem with immunofluorescence is photobleaching , [ 12 ] the fluorophores permanent loss of ability to emit light. [ 1 ]
They were tested in qPCR experiments showing some important differences in the sensitivity and qPCR efficiency, which facilitate the DNA marker selection for analytical purposes. [3] The study concluded that Sybr Green exhibits the highest fluorescence enhancement (AFE) and provides the best characteristic for PCR methods. The second best among ...