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Bookmark or Share- Using NCBI’s Primer-BLAST to design and analyze PCR primersLearn how to use NCBI's Primer-BLAST in our virtual workshop! NCBI's Primer-BLAST combines the primer design features of the popular Primer3 package with a specificity check that uses nucleotide BLAST to search for target and non-target matches in a background database.
- Basic Local Alignment Search Tool - BLASTThe Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.
- Design PCR primers and check them for specificity - National Center for ...Go to the Primer BLAST submission form. Enter the target sequence in FASTA format or an accession number of an NCBI nucleotide sequence in the PCR Template section of the form. If the NCBI mRNA reference sequence accession number is used, the tool will automatically design primers that are specific to that splice variant.
- Primer designing tool - National Center for Biotechnology InformationEnable search for primer pairs specific to the intended PCR template Help. With this option on, the program will search the primers against the selected database and determine whether a primer pair can generate a PCR product on any targets in the database based on their matches to the targets and their orientations.
- Primer-Blast results - National Center for Biotechnology InformationPrimer-BLAST can only find non-specific primers for your template since there are other targets that are similar or identical to your template. You may choose to re-search for specific primers by allowing some of the highly similar off-targets if they are irrelevant to your PCR experiment.
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