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Primer
A primer is a nucleic acid strand, or a related molecule that serves as a starting point for DNA replication. more...
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A primer is required because most DNA polymerases, enzymes that catalyze the replication of DNA, cannot begin synthesizing a new DNA strand from scratch, but can only add to an existing strand of nucleotides.
In most natural DNA replication, the ultimate primer for DNA synthesis is a short strand of RNA. This RNA is produced by an RNA polymerase, and is later removed and replaced with DNA by a DNA polymerase.
Many laboratory techniques of biochemistry and molecular biology that involve DNA polymerases, such as DNA sequencing and polymerase chain reaction (PCR), require primers. The primers used for these techniques are usually short, chemically synthesized DNA molecules with a length about twenty bases. The actual construction of such primers starts with 3'-hydroxyl nucleosides (phosphoramidite) attached to a so-called controlled-pore glass (CPG). The 5'-hydroxyl of the nucleosides is covered dimethoxytrityl (DMT), which prevents the building of a nucleotide chain. To add a nucleotide, DMT is chemically removed, and the nucleotide is added. The 5'-hydroxyl of the new nucleotide is blocked by DMT, preventing the addition of more than one nucleotide to each chain. After that, the cycle is repeated for each nucleotide in the primer. This is a simplified description, the actual process is quite complicated. For that reason, most laboratories do not make primers themselves, but order them by specialized companies.
DNA sequencing is used to determine the nucleotides in a DNA strand. A sequencing method called dideoxy sequencing, also known as chain termination method or Sanger method, uses a primer as a start marker for the chain reaction.
In polymerase chain reaction, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 50 nucleotides (since DNA is usually double-stranded, its length is measured in base pairs. The length of single-stranded DNA is measured in bases or nucleotides), and they match exactly the beginning and the end of the DNA fragment to be amplified. They anneal (adhere) to the DNA template at these starting and ending points, where the DNA-Polymerase binds and begins the synthesis of the new DNA strand.
Primer design
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 is defined as the temperature at which 50% of that same DNA molecule species form a stable double helix and the other 50% have been separated to single strand molecules. The melting temperature required increases with the length of the primer. 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 temperature required to melt it. Melting temperatures that are too high, i.e., above 80 °C, can also cause problems since the DNA-Polymerase is less active at such temperatures. The optimum length of a primer is generally from 20 to 30 nucleotides with a melting temperature between about 55 °C and 65 °C. There are several ways to calculate the melting temperature of primers. (A, G, C and T are the number of nucleotides in the primer, respectively. is the concentration of Na+ in the PCR vial.)
Read more at Wikipedia.org
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