Oligonucleotide primers are necessary when running a PCR reaction. One needs to design primers that are complementary to the template region of DNA. They are synthesized chemically by joining nucleotides together. One must selectively block and unblock repeatedly the reactive groups on a nucleotide when adding a nucleotide one at a time. The main property of primers is that they must correspond to sequences on the template molecule (must be complementary to template strand). However, primers do not need to correspond to the template strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the template DNA strand so elongation can proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches of several nucleotides. Also, both of the 3’ ends of the hybridized primers must point toward one another.
The size of the primer is very important as well. Short primers are mainly used for amplifying a small, simple fragment of DNA. On the other hand, a long primer is used to amplify a eukaryotic genomic DNA sample. However, a primer should not be too long (> 30-mer primers) or too short. Short primers produce inaccurate, nonspecific DNA amplification product, and long primers result in a slower hybridizing rate. On average, the DNA fragment that needs to be amplified should be within 1-10 kB in size.
The structure of the primer should be relatively simple and contain no internal secondary structure to avoid internal folding. One also needs to avoid primer-primer annealing which creates primer dimers and disrupts the amplification process. When designing, if unsure about what nucleotide to put at a certain position within the primer, one can include more than one nucleotide at that position termed a mixed site. One can also use a nucleotide-based molecular insert (inosine) instead of a regular nucleotide for broader pairing capabilities.
Taking into consideration the information above, primers should generally have the following properties:
- Length of 18-24 bases
- 40-60% G/C content
- Start and end with 1-2 G/C pairs
- Melting temperature (Tm) of 50-60°C
- Primer pairs should have a Tm within 5°C of each other
- Primer pairs should not have complementary regions
What makes a good primer?
Here are some guidelines for designing your PCR primers:
- Aim for the GC content to be between 40 and 60% with the 3’ of a primer ending in G or C to promote binding. This is known as a GC Clamp. The G and C bases have stronger hydrogen bonding and help with the stability of the primer. Be mindful not to have too many repeating G or C bases, as this can cause primer-dimer formation.
- A good length for PCR primers is generally around 18-30 bases. Specificity usually is dependent on length and annealing temperature. The shorter the primers are, the more efficiently they will bind or anneal to the target.
- Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other. Because the Tm is dependent on the length, it’s important to keep primers on the shorter end. The bases also impact the Tm, G and C result in higher melting temperatures than A and T. If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
- Typically, 3 to 4 nucleotides are added 5 ’of the restriction enzyme site in the primer to allow for efficient cutting.
- Try toavoidregionsofsecondarystructureandhaveabalanceddistributionofGC-rich and AT- rich domains.
- Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
- Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
- If you are using the primers for cloning, we recommend cartridge purification as a minimum level of purification.
- If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
- If you are using the primers for a PCR reaction to be used in Invitrogen TOPO cloning, the primers should not have a phosphate modification.