What are the criteria to design primers for PCR?

01 January 2017 8 4K Report

Most of the researchers are collecting information from PubMed database. However, I am wondering what are the actual criteria or which points/parameters should I consider to design the primer for my experimental gene?

Laurence Stuart Dawkins-Hall Popular answer

Find attached an SOP detailing optimal primer design and how to screen for things like inter primer annealing (homo dimers); intra primer annealing (hair pin loops) and primer dimers
In essence however consider the following:

Make sure the GC content of your primer is between 40% (no lower) and ~ 60% such that a ~ 20mer has a melting temp of 55C to 65C

Avoid runs of more than 3 consecutive G/C residues and AT residues anywhere in the primer but especially the 3' half of the primer

Make sure the respective Tms of your forward and reverse primer approximating to 55c to 65C are within 2-3C of one another

Pay particular attention to the last 5bp in your primer which confer specificity and efficiency (above all others); In particular:

Make sure the last 5bp do not contain more than 3 x G/C residues

In addition make sure the 3 x GC residues are not at the 3' termini of the primer(s) as this can lead to non specific primer annealing extension;

Inter primer annealing (homo dimers); primer dimers if the opposing primer has a similar GC rich 3' termini. These structures will reduce specific PCR efficiency

Thus, end your primer in a GC GG or CC residue as this confers efficiency and specificity

DO NOT terminate with GGG; GGC; GCC; or CCC

Similarly it is acceptable to end your primer with GT; GA; CT or CA

DO NOT however terminate with 2 x pyrimidine residues, i.e. GAT; CAT' GTT; CTT; GAA or CAA as this can result in inefficient priming/extension

In terms of good primer design complying with the above and situating your primers within a GOI loci simply submit your relevant loci to primer 3 plus:

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/

Alternatively, you can design primers by eye complying with the above and then screen for primer dimers (heterodimers), Tm etc using a variety of freeware tools; May favourite is:

https://www.idtdna.com/calc/analyzer

For intra primer annealing (homo dimers) and hair pin loops the IDT tool will do the job but I find oligo Calculator more user friendly:

http://biotools.nubic.northwestern.edu/OligoCalc.html

Use the 'check self complimentarity feature'

Finally, another feature of oligo calculator is BLAST:

You should ALWAYS BLAST your primer sequences to ensure they are specific, i.e. only bind the full length of the primer to just your target region:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

Having submitted your primer make sure you get 100% 'identity' for the full length ('query') of your ~ 21mer primer sequence

Manoj Pohare

Designing primers for PCR amplification also depends on the method of cloning you are going to implement, like restriction enzyme based, ligation independent cloning, recombination based cloning, etc.

You can find many primer designing rules online. Importantly less GC content towards 3' end of primers, as it will reduce the chance of mismatch.

S M Shamsul Islam

There is a lot of thigs we have to remind before to do primer design. Most of the time just put the sequence in NCBI primer blast and pick up primer is not correct. you have to consider what is your gene size, where is the location of the primer, the promoter region, the start and stop codon.

Actually depending on your work there are several criteria.

Laurence Stuart Dawkins-Hall

Find attached an SOP detailing optimal primer design and how to screen for things like inter primer annealing (homo dimers); intra primer annealing (hair pin loops) and primer dimers
In essence however consider the following: