Primers are essential for initiating DNA amplification, whether for the purposes of detection, cloning or sequencing. Thus, it is crucial to understand how to successfully design primers. Here are the four tips for efficient primer designand primer management.

Understand the Basic Primer Design Rules

The primer you design impacts the entire DNA amplification process. DNA polymerases, the enzymes that catalyse DNA replication, can only initiate the replication process by adding nucleotides to primers. In order to produce the desired DNA sequence, you must start with the right primer. Thus, proper primer design is necessary for successful DNA amplification. Here are 14 basic guidelines for constructing primers:

• Primers are always specified 5' to 3', left to right. Verify that your primers are designed and ordered in the correct orientation.
• Primers for PCR and sequencing should be between 18 to 25 nucleotides in length.
• Primers for PCR and sequencing should have a GC content between 40 and 60%, with the 3′ of a primer ending in C or G to promote binding.
• The 3' end of the primer should be an exact match to the template DNA, because extension by DNA polymerase, during PCR, depends on a good match at the 3' end.
• In the last 5 bases at the 3′ end of the primer, make sure that there are at least 2 G or C bases (GC clamp). G-C base pairs have a stronger bond than A-T base pairs (3 hydrogen bonds versus 2).
• Where a restriction site has been added onto the end of a primer, typically, 5-6 nucleotides are added 5’ of the restriction enzyme site (aka a “leader sequence”) in the primer to allow for efficient cutting.
• Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT) as this can cause primer mispriming.
• Avoid regions of secondary structure; namely 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/hairpins or primer-dimers instead of annealing to the desired DNA sequences. Thus have a balanced distribution of GC-rich and AT-rich domains. Toolslike IDT Oligo Analyzer can help you to detect secondary structure.
• In general, the ΔG value for dimer analysis should be between 0 to −9 kcal/mole for optimal design. Values more negative than this may adversely affect PCR reactions.
• For hairpins, the melting temperature, (Tm ) of the hairpin should be lower than the annealing temperature for the reaction; on average it should range between 55°C and 65°C. The Tm for the strongest hairpin should be at the very least 50°C and below the annealing temperature. 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.
• Primer pairs should have similar Tm’s with a maximum difference of 5°C and should not be complementary to each other.
• If you are using the primers for mutagenesis, try to have the mismatched bases towards the middle of the primer.
• Verify your primers’ specificity so they won’t bind to other genomic regions through NCBI Primer BLAST or UCSC in-silico PCR.
• In order to verify the Tm, try an annealing temperature gradient PCR reaction to find the optimal Tm according to your primer and enzyme.

Dear Dr.Rostami,

Hello and Thanks for your response;

Yes, I know all of rules that is needed for designing primers but, these primers are designed to create a new sequence by PCR technique to do the next purposes. So, it is unavoidable.

Hope you have some SNP's in templates. so order mixture of nucleotide for those two points only. Then you can amplify both alleles.@faezeh Babaei

can you design a primer so that the mismatches are nearer the middle of the primer. If not you will have to run a gradient of annealing temperatures for your pcr starting at about 10c below the annealing temperature of the mismatched primer or the lower temperature annealing primer whichever is the lower

Make your forward primer longer by adding more bases to the 3' end so that the mismatches are closer to the center. Or just try them as made with a gradient of annealing temps to see if they do what you need. I've had strong amplification from primers that had 2 bp of mismatch at the terminal 3' bases.

I agree with Paul Rutland and Katie A Burnette. Increasing the size of mismatched primer is a good way to equilibrate the annealing temperature close to reverse primer. You can also search the secondary PCR targets for your primer using 'primer blast' tool. This will help you to reduce the possibility of producing nonspecific product in your PCR.