A primer is a specifically designed sequence of bases approximately 18-20bp in length. Primers allow DNA polymerase to start elongating DNA. Primers are used in the Polymerase Chain Reaction (PCR) as a step in amplifying a short sequence of DNA. They are unique as they only bind to particular region in DNA where DNA transcription starts off from 5' to 3'. As primers are widely used in polymerase chain reaction (PCR), this characteristic of primers is advantagenous in preventing contamination from different DNA from other sources and specific region of desired DNA can be amplified.
A primer is a short string of nucleotides used in the synthesis of DNA. Primers are essential for the function of DNA polymerase as the enzyme can only build nucleotides onto an existing DNA strand. They are synthesised by an enzyme called Primase and are complementary in bases to the single stranded DNA template with a 3' OH end . Primers are short RNA molecules in living cells, however, DNA serves as the primer in non-living cells (in vitro) such as in PCR . DNA polymerase I plays an important role in the replication process by the removal of the RNA primer during its proof reading activity . The PCR procedure requires 3 different temperatures for the 3 crucial steps to occur. firstly the DNA strands have to be denatured to form single strands at 94°C, secondly the cooled single stranded DNA are annealed to primers at 55°C and finally the synthesis and extension of the newly formed DNA strands using TAQ polymerase at 74°C. Different primers have slightly different annealing temperatures. The syringency and specificity of PCR recations depend on the primer used and the concentartion of Mg2+ to depict accurate annealing tempearture, this allows the correct PCR product to be formed.
Primers are between 18-30 bases depending on the genome, the longer the genome you are intending to clone, the longer the primer. The longer the primer, the more mismatches it can tolerate. The melting temperature needs to be around 60-65°C as this is the annealing temperature for the primers in the PCR. This is dependent on the nucleotide bases used and the length of the primer and can be calculated by the following equation:
Tm= (4xG/C) + (2xA/T)
G or Cs at the 3' end of the primer are necessary as these are stable bases and don't tend to mismatch as easily as a T and A. Secondary structures in the primer and DNA target such as stem loops or bulges should be avoided as these would prime the target DMA sequence poorly.
It is also important ot avoid primer hybridisation as this would cause self-amplification of primers. This is termed Primer Dimer.
Computational approaches are now used for optimum primer design.
- ↑ Berg, J. et al., (2007), Biochemistry, 7th Edition, New York, WH Freeman. Page 852
- ↑ Hartl D. and Jones W., (2009), Genetics; Analysis of Genes and Genomes, 7th edition, Sudbury, Jones and Bartlett publishers. Page 56
- ↑ Berg, J. et al., (2007), Biochemistry, 7th Edition, New York, WH Freeman. Page 864