Hi, I have few primers that are previously reported to amplify a conserved region. The ambiguity is that I want to be sure if they are correctly reported before ordering the same set of primers. How can I check their authenticity? How do I use primer blast ?
To check a previously reported pair of primers for specificity, you can use the Primer BLAST tool provided by the National Center for Biotechnology Information (NCBI). Here is a step-by-step guide on how to do this:
Access Primer BLAST Submission Form:
Go to the Primer BLAST submission form on the NCBI website.
Enter Primer Sequences:
Enter the primer sequences that you want to check in the Primer Parameters section of the form. If you only have one primer sequence, you will also need to provide a template sequence.
Select Specificity Checking Parameters:
In the Primer Pair Specificity Checking Parameters section, choose the appropriate source organism and select the smallest database likely to contain your target sequence for precise results. For broader coverage, you can choose the nr database without specifying an organism.
Submit Search:
Click on the “Get Primers” button to submit your search and retrieve information on template and specificity.
Review Results:
The tool will provide you with information on primer specificity based on the parameters you set. It will indicate if there are any potential off-targets or non-specific binding sites for your primers.
By following these steps using Primer BLAST, you can effectively check a previously reported pair of primers for specificity and ensure they are suitable for your PCR experiment.
According to my knowledge and believe you may follow the steps:
To ensure the authenticity and accuracy of your primers before ordering them, you can use the Primer-BLAST tool provided by the National Center for Biotechnology Information (NCBI). Primer-BLAST allows you to check your primers against a database of sequences to verify their specificity and efficiency. Here’s a step-by-step guide on how to use Primer-BLAST:Access Primer-BLAST: Go to the NCBI Primer-BLAST webpage: Primer-BLAST.Input Primer Sequences:In the "Primer Parameters" section, enter the forward primer sequence in the "Forward Primer" field.Enter the reverse primer sequence in the "Reverse Primer" field.Specify PCR Template:Enter the template sequence (the target DNA sequence) or the accession number of the sequence you are interested in amplifying. You can also input a sequence directly if you have it in FASTA format.Set Search Parameters:In the "PCR Product" section, you can specify the expected product size range if you know it.You can also adjust parameters such as primer melting temperature (Tm), GC content, and more in the "Primer Pair Specificity Checking Parameters" section.In the "Database" section, select the appropriate database to check against (e.g., "Refseq mRNA," "Refseq genomic," etc.).Run the Search:Click the "Get Primers" button to initiate the Primer-BLAST search.Analyze Results:Primer-BLAST will provide you with results indicating the specificity of your primers. Look at the "Summary" section to see if your primers match the intended target sequences.Check the "Detailed Results" section to see any potential off-targets and the location of the matches within the sequences.Pay attention to any warnings about primer-dimer formation or secondary structures that could affect the efficiency of your PCR.Verify Conserved Region:If your primers are supposed to amplify a conserved region, ensure the results show successful amplification across different sequences representing the conserved region.You might also want to run the primers against a multiple sequence alignment of the conserved region to visually confirm the binding sites.
I think I did not specify that the sequence that I am trying to amplify is from a conserved sequence ( derived after studying several viral isolates from different regions of our country using clustal W). So I wont be able to select any organism or species in NCBI to match against. But what I instead did was use the individual primers and match them randomly on NCBI to check whereit is matching. It's actually matching with the genome region that I want of the virus. Thank you all for your recommendations. Really appreciate it. Muhammad Umar Tesfaye Humnessa
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