We ordered a primer for our project. I want to test whether the primer amplifies genes from other organisms too or not. Is there any software to test whether my primer works for other genomes or not? I have attached the primer sequence.
You can blast your primer sequences against the target organism. I hope that could give you an idea whether your primers is going to hybridize with the genome or not. But to get an amplification that is not the only criteria, it also depends on which region of genome it is hybridizing in order to give amplified products. Best way to do it by performing PCR with the genomic DNA.
As more and more genome data becomes available Primer-Blast should be your first step. Consider which species might be present in your sample to make a selection in Primer-Blast to blast against. This will give you already a very good indication.
If you want to avoid aspecific primer binding, make them a little bit longer, avoid stretches of mono- or dinucleotide repeats and average the GC% of the primers towards the GC% of the genome.
If genomic data available then best way to test the specificity of your primer is to run blast it will give you matches present in your genomic DNA. To avoid the nonspecific binding you need to increase the annealing temperature of your primer. FOr this purpose your you increase the primer length and select the primer which is having more CG content.
Indeed, higher Ta might help.
Increasing the annealing temperature depends on the Tm of your primers and will only avoid aspecific amplification in case primers bind to not fully complementary sites.
To avoid amplification in non-target genomes you'll have to anticipate by designing good specific primers, using the tips mentioned above.
Thank you for all of your response. I tried running in silico PCR with different target organisms but i found zero match. While performing primer blast using NCBI tool, i obtained positive hybridization results but with the forward primer only. I am new to using bioinformatics tool and maybe i did something wrong. I was searching if there were any in silico PCR tools that allows us to input a specific target sequence because the tool i found only allowed me to test run my primers within a specific set of organisms. and moreover, the primer we ordered was based on a journal article.
This link I hope it is useful for you.
https://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers/
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