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A strip of eight PCR tubes, each containing a 100 μL reaction mixture Placing a strip of eight PCR tubes into a thermal cycler. The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study.
Chimeric polymerases overcome many limitations of native enzymes and are used in direct PCR amplification from cell cultures and even food samples, thus by-passing laborious DNA isolation steps. A robust strand-displacement activity of the hybrid TopoTaq polymerase helps solve PCR problems that can be caused by hairpins and G-loaded double ...
The staggered extension process (also referred to as StEP) is a common technique used in biotechnology and molecular biology to create new, mutated genes with qualities of one or more initial genes. The technique itself is a modified polymerase chain reaction with very short (approximately 10 seconds) cycles. In these cycles the elongation of ...
In real-time PCR with dsDNA dyes the reaction is prepared as usual, with the addition of fluorescent dsDNA dye. Then the reaction is run in a real-time PCR instrument, and after each cycle, the intensity of fluorescence is measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). This method has the ...
Second, the formerly obtained PCR products are combined together into the overlap extension PCR reaction, where the complementary overhangs bind pair-wise allowing the polymerase to extend the DNA strand. Eventually, outer primers targeting the external overhangs are used and the desired DNA product is amplified in the final PCR reaction.
In the laboratory setting, Pfu is used to amplify DNA in the polymerase chain reaction (PCR), where the enzyme serves the central function of copying a new strand of DNA during each extension step. It is a family B DNA polymerase. It has an RNase H-like 3'-5' exonuclease domain, typical of B-family polymerase such as DNA polymerase II. [1]
The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles. The primer will anneal at the highest temperature which is least-permissive of nonspecific binding that it is able to tolerate.
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.