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Primer

From Molecular Biology Wiki

A primer is a nucleic acid strand, or a related molecule that serves as a starting point for DNA replication. 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; the chain termination method (dideoxy sequencing 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. [Na+] is the concentration of Na+ in the PCR vial.)

  • "GC"-method : Fast and simple, for primers with more than 13 nucleotides.
    <math>T_\mbox{m}=64+41 \cdot \frac{G+C-16.4}{A+G+C+T}</math>
  • "Salt-adjusted"-method : More accurate than GC, for primers with more than 13 nucleotides.
    <math>T_\mbox{m}=100.5+41 \cdot \frac{C+G}{A+C+G+T}-\frac{820}{A+C+G+T} \cdot 16.6 \cdot \log_{10}([\mbox{Na}^+])</math>
  • Base-stacking calculation : Most accurate, but complicated
<math>T_\mbox{m}=\frac{\Delta H \frac{\mbox{cal}}{\mbox{mol}}}{\Delta S+R \ln(\frac{primer}{2})}-273.15 \ ^\circ \mbox{C}</math>
where
<math>\Delta H</math> is the enthalpy of base stacking interactions adjusted for helix initiation factors
<math>\Delta S</math> is the entropy of base stacking adjusted for helix initiation factors and for the contributions of salts to the entropy
<math>R</math> is the universal gas constant <math>\left (\frac{1.987\mbox{ cal}}{\mbox{mol} \cdot ^\circ \mbox{C}} \right)</math>

The free software package primou can calculate the annealing temperature according to the base stacking method.

Also, a primer should not easily anneal with itself or others of its kind, building loops or hairpins in the process. This could hinder the annealing with the template DNA. However, small hairpins are usually unavoidable.

Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid isoleucine might be "ATH", where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine, according to the genetic code for each codon. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR.

Degenerate primers are widely used and extremely useful in the field of microbial ecology. The allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations.


===Degenerate primers=== Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid isoleucine might be "ATH", where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine, according to the genetic code for each codon, using the IUPAC symbols for degenerate bases. Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR.

Degenerate primers are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations.

References

An earlier version of the above article was posted on Nupedia.

External links

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