PCR Primers
Definition of a PCR Primer:
PCR Primers are short single-stranded, synthetically synthesized oligonucleotides usually shorter than 50 nucleotides (often 18-25 nucleotides).
Applications of PCR Primers
PCR primers are important as they are complementary to the beginning and end of the DNA fragment of interest which one needs to amplify. Primers therefore select the boundaries of the region to be amplified by PCR. During the PCR annealing cycle, PCR primers anneal to the complementary region of the DNA. DNA polymerase binding and the 3’ OH of the oligo allows the synthesis of DNA to occur.
Choosing PCR Primers
Important Factors To Consider When Choosing Primers:
- Optimal Tm (annealing T) ~ 50-60°C
- Oligonucleotide GC-content should be between 40-60%.
- Calculated Tm (melting temperature) for both primers used in reaction should not differ >5°C.
- Primer annealing temperature is usually 5°C below the calculated lower Tm. However it should be chosen empirically for individual conditions.
- Inner self-complementary hairpins of >4 and of dimers >8 should be avoided.
- 3' terminus is extremely case sensitive - it must not be complementary to any region of the other primer used in the reaction and must provide correct base matching to template.
- If possible, primers should start and end with 1-2 G or C residues
- Primers should NOT have any extendisve secondary structure or self-complementarity (can result in Primer-dimers).
PCR Primer Design
Programs to assist you in PCR Primer Design:
Primer3
WebPrimer
Find them here - PCR Primer Design Tools.