I am working on primer designing and had the following questions
1. which primer design software is the best primer quest or NCBI primer blast
2. When I design a primer on primer quest, I see Tm difference e.g a primer has 62 tm on primer quest , but it has Tm on NCBI blast 59.8, same if a primer has Tm 61 on NCBI blast, but it has 65+ on Primerquest oligo Analyzer
3. Normally I am using mRNA gene for primer designing, either I need to select "Primer must span exon-exon junction" or no preference, but I always tick " Primer pair must be separated by at least one intron on the corresponding genomic DNA " and intron length MIn 200, Max 1000000 (default), Please also tell me the advantages of span exon-exon junction
I know my question is too long, but I am confused in the above things please guide me
Thanks in Advance
Hi Muqeet, I agree with Sebatian, best is a very subjective judgement. Having the amplicon span a exon-intron-exon junction where the intron is large, makes the likely hood of amplifying product from any remaining genomic DNA less likely. Relatively small Tm differences can be due to use of different equations used to calculate the Tm, but also may be due to different assumptions of mono and divalent cation concentrations in the PCR reaction.
Frank
For the 1st question, I used BLAST for all primer designing which works better for Traditional PCR, but for Real time PCR, I used the primer designing tool from IDT which enables to detect which technique to be used and it will be useful for probe designing as well.
For the second question, it depends on the calculation equation for Tm value, usually it will be mentioned in the Kit that you are going to use, so it's better to calculate it manually.
For the 3rd question, it depends on your stating material, if you will use the RNA, then exon-exon junction is better to get rid of all the genomic DNA, and it's not necessary to use this feature for the designing of both primers as you know that real time PCR is working only for short DNA targets (max. 200 nt). If you are looking for a mutation in the intron then you have to specify the stating and ending nucleotide on the reference gene that you are using, which is available in BLAST.
To avoid amplification of contaminating genomic DNA, you should design primers so that one half of the primer hybridizes to the 3′ end of one exon, and the other half to the 5′ end of the adjacent exon. To do this, you should select “Primer must span an exon-exon junction.”
Also, regarding different software usage for primer designing, it doesn’t matter which software you are using since different software has different algorithms. In my experience, eventually, the final TM written by the company on the primer tubes is different from what we have calculated using different software. Thus, the important thing is that you should consider and follow main factors such as GC content and the proper difference between the Tm of two primers (reverse and forward).
To design a primer for real time, please consider these guidelines:
1- https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwirwN2D8czrAhVSoXEKHR8iCgYQFjABegQIBBAB&url=https%3A%2F%2Fwww.qiagen.com%2Fjp%2Fresources%2Fdownload.aspx%3Fid%3Dd6191d0e-701b-4eb1-bafa-d7ab7677875f%26lang%3Den&usg=AOvVaw1SYvdE_MSOUFRhcIwmSUv6
2- https://bitesizebio.com/10041/designing-qpcr-primers/
Good luck!
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