i have designed a primer but i found it gives 200 hits over than the intended sequence (5 of them in the organism that i work with). this give poor pcr results. i want to avoid this but i don,t know the way
Hello Noha,
The only thing I can advise you with is to design a new primer that anneals slightly upstream or downstream of the genonic region where your current primer does, bearing in mind the "rules" for primer designing (for instance: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html )
Beware that, depending on the PCR variant, these rules may differ in some aspects!
Hope it helped,
João Nina
Hi, which species you are working with? I suggest to make the multiple alignments of the 5 sequences that are the most similar to your target sequence in your organism. Then identify the places where you find nucleotides that are specific fro your target gene and design the primers in the areas where you found more specific nucleotides. There are other important aspects as the location of the specific nucleotides in the 3' end to increase the primer binding specificity.
For a better understanding, it is better you check this paper that explains a tool for designing sub-genome specific primers in wheat Article GSP: A web-based platform for designing genome-specific prim...
you can even consider to use this tool to perform the multiple alignment of your sequences and the the software is able to suggest you some primers pairs adequate for your target sequence. I hope this helps.
The problem appears like you dont know how to design primers. Please follow the previous advice and also try to learn to design the primer designing. Ask your colleagues to help.
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