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Other molecular items would be required Ions, magnesium, dNTP, primers, and buffer.
What other molecular items would be required?
Polymerase chain reaction (PCR) is a technique for obtaining a large number of copies of a particular DNA fragment from a small amount of sample. It is sufficient to start from a single copy of that original fragment, or mould. This technique is based on the natural property of DNA polymerases to replicate strands of DNA, using alternating high and low-temperature cycles to separate the newly formed strands of DNA from each other after each replication phase, and then allow the strands of DNA to rejoin so that they can be duplicated again. Today, the entire PCR process is automated by means of a device called a thermal cycler, which allows the reaction tubes to be heated and cooled in order to control the temperature required for each stage of the reaction.
The common PCR is performed with cycles that have three temperature steps:
- DNA denaturation double strand
- Hybridization of the primers to the specific zone 3'of each of the strands
- Extension of the primer by action of DNA polymerase
The components necessary to carry out a PCR are:
Divalent Ions: necessary for polymerase function, magnesium ions are generally used and are in the form of magnesium chloride salt MgCl2 . The final concentration of Mg++ should be between 1.5-2.0 mM and is optimal for most PCR products.
dNTP: deoxyribonucleotides triphosphate, used by the polymerase to extend the chain.
Primers: are two short nucleotide sequences that are complementary to the DNA fragment mold. They are generally constituted between 20-40 nucleotides on average and with a GC content of 40-60%.
Buffer: Solution necessary for the reactions to take place, it is in charge of keeping the pH stable.
Taq polymerase: Enzyme in charge of DNA synthesis by adding dNTP. It supports high temperatures however has no corrective activity.
All these components are mixed in one solution.
Thus, Ions, magnesium, dNTP, primers, and buffer.
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