Enter an organism name (or organism group name such as enterobacteriaceae, rodents), taxonomy id or select from the suggestion list as you type. Help Entrez query (optional) Help Primer specificity stringency Primer must have at least 1 2 3 4 5 6 total mismatches to unintended targets, including
Try "re-search for specific primers" feature on Primer-BLAST result page. When only non-specific primers are found, the result page offers the option to re-search for specific primers.
The 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.
We describe a robust and fully implemented general purpose primer design tool that designs target-specific PCR primers. Primer-BLAST offers flexible options to adjust the specificity threshold and other primer properties.
To find primers for a group of related sequences, Primer-BLAST aligns the longest sequence to the rest to find common regions. It uses these to limit the locations of primers. The longest sequence is also used as the representative template sequence in the results.
The good news is that an existing tool, Primer-BLAST, fills in nicely for the functions of both Forward and Reverse e-PCR, and has the additional benefit of de novo primer design.
Primer design is a crucial initial step in any experiment utilizing PCR to target and amplify a known nucleotide sequence of interest. Properly designed primers will increase PCR amplification efficiency as well as isolate the targeted sequence of interest with higher specificity.
Sanger sequencing of the purified PCR products is performed using BigDye Terminator v3.1 cycle sequencing with 3 μL of DNA template and 1 μL of 2 μM of each primer.
The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain.
