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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.
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. Real-time PCR can be used ...
Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple sequences at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform.
In research or diagnosis DNA amplification can be conducted through methods such as: Polymerase chain reaction , an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA by polymerizing nucleotides, a concept which is applicable to numerous fields in modern biology and related sciences.
Several DNA polymerases have been described with distinct properties that define their specific utilisation in a PCR, in real-time PCR or in an isothermal amplification. Being DNA polymerases, the thermostable DNA polymerases all have a 5'→3' polymerase activity, and either a 5'→3' or a 3'→5' exonuclease activity.
An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted. [4]
Although PCR is usually associated with thermal cycling, the original patent by Mullis et al. [2] disclosed the use of a helicase as a means for denaturation of double stranded DNA thereby including isothermal nucleic acid amplification. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that ...
The PCR method is extremely sensitive, requiring only a few DNA molecules in a single reaction for amplification across several orders of magnitude. Therefore, adequate measures to avoid contamination from any DNA present in the lab environment ( bacteria , viruses , or human sources) are required.
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