Hi! I am currently designing primers for the human UGT1A1 gene using the Primer3 software. However, when I BLAST the primers against the Human RefSeq database, I get significant matches with other human genes, no matter what combination I try. Is there any solution to decide a suitable primer?What is the optimal range for the parameters in BLAST result that I can use to decide the best primer? Or should I relax any other input parameters in the Primer software tool?
Hi! You must design primers in software and selects several them base on primer design parameters. Then blast them and select one of them that have best blast result.
Dear Dhanya
The specificity of primers depends on several parameters: annealing temperature, length of the primer sequence, the GC content of the primer sequence etc.
My experience is:
- the longer your primer sequence the more specific the binding
- the higher the annealing temperature the more specific the binding
In practice this means:
- try to find primer sequences of at least 20 nucleotides (but not more than 23-24 otherwise the pcr conditions will be too difficult): 20, 21, 22nu would be fine
- the GC content of the primer sequences should be between 40-70%. Not less and not more!
- the longer the primer sequences and the higher the GC content the higher will be the annealing temperature
- In summary, select such primer pairs with 20-22nu (if not other possibility 23nu), a GC content 40-70% (better 50-70%), and an annealing temperature of more than 55°C (up to 60-61°C).
If you find suitable, specific primer sequences (after checking agains the human RefSeq database as you did), don't forget that you have to check the new primers in a pcr gradient applying several temperatures to check which temperature gives best specific products!
Good luck!
Carolina
Adding to Carolina's comments on parameters, it is usually good to avoid many G or C bases at the 3'-end of your primer. To combine good stability with high specificity a primer should ideally be GC rich in the 5'-end but less so in the 3'-end.
To avoid primer annealing to closely matching targets try to make sure that a mismatch would be close to the 3'-end. In stringent conditions this should minimize undesired extension.
Also you can compare the predicted amplicon sizes between your desired target with the significantly matching targets. If any of your UTG1A1 primer pairs produce a shorter amplicon than those of the matching genes they would give a competetive advantage, especially using relatively short annealing and extension times.
Standard primers are inexpensive, so I would suggest you try a few pairs and combinations. Subtle differeces can have a big impact. I usually pick at least two F and two R primers and look which of the combinations works best.
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