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Primer-blast tries to find target-specific primers by placing candidate primers on unique template regions that are not similar to other targets.
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. If one or both primer ...
Jun 18, 2012 · Primer-BLAST is the only program among the three that will place primers on different exons (i.e., to span an intron) to avoid amplification of genomic DNA and also the only one to allow customization of the number of nucleotide matches on either side of an exon/exon junction.
Nov 28, 2023 · 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.
One needs to design primers that are complementary to the template region of DNA. They are synthesized chemically by joining nucleotides together. One must selectively block and unblock repeatedly the reactive groups on a nucleotide when adding a nucleotide one at a time.
Feb 4, 2022 · 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.
Oct 14, 2022 · Primer-BLAST is a combination of Primer3, a popular primer designing tool, and NCBI BLAST . Primer-BLAST has two basic modes. It can: Design primers to specifically amplify a template sequence or a set of related templates from a background nucleic acid population (BLAST database).
Jun 18, 2012 · Primer-BLAST allows users to design new target-specific primers in one step as well as to check the specificity of pre-existing primers. Primer-BLAST also supports placing primers based on exon/intron locations and excluding single nucleotide polymorphism (SNP) sites in primers.
Nov 7, 2022 · Video Recording: Using NCBI’s Primer-BLAST to Design and Analyze PCR Primers. Keyboard controls: Space bar - toggle play/pause; Right and Left Arrow - seek the video forwards and back; Up and Down Arrow - increase and decrease the volume; M key - toggle mute/unmute; F key - toggle fullscreen off and on.
Primer-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.
