Using NCBI’s Primer-BLAST to Design and Analyze PCR Primers

Duration: 2 hours

Content Difficulty: Intermediate

Target Audience:

This workshop is designed for people who need to design PCR primers to amplify target sequences or to who need to predict the amplified products and the specificity of designed primers. 

Workshop description:

Biological researchers often design specific PCR primers to amplify a single genomic or mRNA template or a set of closely related templates. In addition, PCR amplification with specially designed primers is sometimes used to identify an organism or group of organisms based on targeted RNA or genomic DNA amplification of an isolate. 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. Primer-BLAST primers are suitable for use in all PCR based molecular biology protocols including target identification / verification, cloning, variant analysis, and gene expression.

In this workshop, you will learn to:

• Design primers specific for mRNA (cDNA) templates that will not cross amplify genomic DNA.
• Design primers for specific genomic templates such as an exon of a gene.
• Identify potential products for a pair of primers.
• Interpret primer alignments on intended and off-target matches to pick the best primers.
• Choose the best background BLAST database to check primer specificity or identify targets.
• Display Primer-BLAST results in the context of the template or genome using graphical displays.