Can NCBI blast highlight the specificity of a primer pair for PCR amplification of a desired sequence. What would be the best possible ways to check the primer specificity?
You should check these for primer specificity:
1. whether or not your primer pairs are unique, they won't bind to other locations in the genome except your intended gene or DNA fragment.
2. will primer pair bind to each other (forming primer dimer)-- (1) self-dimer or (2) hetero-dimer
3. the possibility of the forming of secondary structure of the primers, which may cause difficulties for PCR amplification
4. Tm mismatch-- the Tm of two primers should be designed to close to each other
5. the annealing temperature (Ta)-- One consequence of setting Ta too low (much lower than Tm) is that one or both primers will anneal to sequences other than the intended target and lead to non-specific PCR amplification
6. Except NCBI, other web-based primer analyzing tools are also available, such as the 'OligoAnalyzer 3.1' program from IDT company.
http://www.idtdna.com/calc/analyzer
Hi, you can use NCBI blast, to design your specific primers, if the gene is sequenced and registered in NCBI, just you make the reference and push pick primers to pick yours, usally the on line software, gives 5 poissibilities of specific amplicon with a determined size, and always the best pairs, as fidelity, are the first, and if the gene wasn't registered on NCBI, you can enter manually the sequence, but be careful to don't forget or delet by mistake some bases, and after pick your primers, it's a good way to be sure about the specificity of the primers, but you couldn't be absolutely sure, before that you run the electrophoresis gel, after that you run your PCR. good work.
You should check these for primer specificity:
1. whether or not your primer pairs are unique, they won't bind to other locations in the genome except your intended gene or DNA fragment.
2. will primer pair bind to each other (forming primer dimer)-- (1) self-dimer or (2) hetero-dimer
3. the possibility of the forming of secondary structure of the primers, which may cause difficulties for PCR amplification
4. Tm mismatch-- the Tm of two primers should be designed to close to each other
5. the annealing temperature (Ta)-- One consequence of setting Ta too low (much lower than Tm) is that one or both primers will anneal to sequences other than the intended target and lead to non-specific PCR amplification
6. Except NCBI, other web-based primer analyzing tools are also available, such as the 'OligoAnalyzer 3.1' program from IDT company.
http://www.idtdna.com/calc/analyzer
These are good suggestions -- I'd just like to add the specific link to the "Design Tool", which is integrated with blast and does all these things for you automatically. http://www.ncbi.nlm.nih.gov/tools/primer-blast/
This is a challenging - and very necessary - exercise, and I shall be trying all the software suggestions that turn up in these discussions.
What you need to do is to identify all sequences in the database that have homology to both forward and reverse primers, with the hit sequences placed so that they can actually form a PCR product.
My best method at present is to perform a blast search for each primer individually, excluding the target taxon, copy the list of accession numbers, sort it with a word processer, then compare the lists. Excel could probably do this quite efficiently if you're an excel wizard.
Any accession number that occurs in both lists needs to be investigated as a potential cross-reacting sequence. Then you have to consider the positions of any mismatches, whether or not they are destabilising, and the position and orientation of the hits in the sequence.
If you get hundreds of candidate cross-reacting sequences, you can probably assume that your primers are non-specific. At this point It will be easier to design new primers than to check each of the candidates in detail.
Hi, Andrew Jenkins
That is what I am doing. I am wondering if there are better methods.
BTW, how to reply to a particular post in a thread?
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
insert your primers in primer parameters section and select your organism and click on get primer to analyzed your primers
There are many websites for creating the primers that already you got the answer. However I would like to recommend the primer analysis software that is free and easy to use. After creating the primers and before order I always test them in Netprimer (please find below the link):
http://www.premierbiosoft.com/netprimer/index.html
I have constructed a pipeline to design primers and check the specificity. Hope it can give some hint.
Guo L, Yang Q, Yang JW, et al. (2020) MultiplexSSR: A pipeline for developing multiplex SSR‐PCR assays from resequencing data. Ecology and Evolution 10, 3055-3067.
Article MultiplexSSR: A pipeline for developing multiplex SSR‐PCR as...
Hi
Primer blast works only a specificity check when a target template and both primers are given. In the primer Pair specificity checking Parameters section, select the appropriate source organism and the smallest Database that is likely to contain the target sequence. These settings give the most significant results.
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