Polymerase chain reaction
Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. Like amplification using living organisms, the technique allows a small amount of DNA to be amplified exponentially. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations. PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing. PCR in practice
PCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or a non-coded sequence. PCR typically amplifies only short DNA fragments, usually up to 10 kilo base pairs (kb). Certain methods can copy fragments up to 25 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell - for example, a human cell contains about three billion base pairs (3 Gbp). PCR, as currently practiced, requires several basic components . These components are: * DNA template, which contains the region of the DNA fragment to be amplified * Two primers, which determine the beginning and end of the region to be amplified (see following section on primers) * Taq polymerase (or another durable polymerase), a DNA polymerase which copies the region to be amplified * Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA * Buffer solution, which provides a suitable chemical environment for the DNA Polymerase * Divalent cation, magnesium or manganese ions
* Monovalent cation, potassium ions
The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. The lid of the thermal cycler is heated to prevent condensation on the inside of the reaction tube caps. Alternatively, a layer of oil may be placed on the reaction mixture to prevent evaporation. Typical reaction volumes range from 15-100 Î¼l. Primers
The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands â€” often not more than 50 and usually only 18 to 25 base pairs long â€” that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand. The choice of the length of the primers and their melting temperature (Tm) depends on a number of considerations. The melting temperature of a primer -- not to be confused with the melting temperature of the template DNA -- is defined as the temperature at which half of the primer binding sites are occupied. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the maximum temperature allowed to be applied in order to melt it, as melting temperature increases with the length of the primer. Melting temperatures that are too high, i.e., above 80Â°C, can cause problems since the DNA polymerase is less active at such temperatures. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55Â°C and 65Â°C. The above mentioned considerations make primer design a very exacting process, upon which product yield depends: * GC-content should be between 40-60%.
* Calculated Tm for both primers used in reaction should not differ >5Â°C, and Tm of the amplification product should not differ from primers by >10Â°C (In practice may not be true always)....
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