Primer Design

Beacon Designer

Beacon Designer Software supports the design of SYBR Green primers, TaqMan Probes, Molecular Beacons, HRMA Primers, NASBA Assays, FRET Probes and Scorpions. All design is automated with optimal parameters set for specific assays. One of the advantages to this software is that it is possible to BLAST sequences and search for template structures from within the software.  The significance of this is that both of these searches are used during the design process so that regions exhibiting significant cross homology and template structures are avoided.  You can also design primers over junctions to avoid amplifying genomic contamination.

An online manual is not currently available; however, guides can be accessed through the software.  The software is located on two PCs in the Core Computer Lab, Room F6-27.

For a more thorough overview of the software, visit Premier Biosoft’s website.

Primer Design Considerations:
  • Keep in mind that primers designed for end-point PCR may not be optimal for real-time PCR.
  • Primer design is an important consideration for Real-Time PCR. Primers should be highly specific to your sequence of interest.  Blast your primers to check for specificity. 
  • Check primers for secondary structures, such as self-dimers and hairpins. As a general rule, the ΔG value for these structures should be more positive than -9.0 kcal/mole.
  • Order validated pre-developed primers from places like IDT, when possible.
  • If designing your own primers, use a software specific for qPCR and the chemistry (SYBR Green or TaqMan) you are using. Beacon Designer is available in the Core Computer Lab and the NCBI tool Primer BLAST can be accessed online.
    • It is good practice to design and order several pairs for each sequence of interest. Test them and choose the best one.
    • If you are having trouble finding the accession number that correlates to your gene, go to the IDT pre-developed assay page, query for your gene and retrieve the accession number from the returned results.
  • When possible, design your primers to span an exon-exon junction. This design reduces the risk of false positives from amplification of any contaminating genomic DNA, since the intron-containing genomic DNA sequence will not be amplified.
  • Consider the following when designing primers for SYBR Green Chemistry:
    • PCR product/amplicon size (80 to 150 is optimal).
    • The optimal melting temperature (Tm) of your primers is 60 to 64˚C. Ideally, the melting temperatures of the 2 primers should not differ by more than 2˚C so that both primers bind simultaneously and efficiently amplify the product.
    • The annealing temperature (Ta) is dependent upon the length and composition of your primers. In general, this temperature should not be more than 5˚C below the Tm of your primers.  If the Ta is too low, one or both primers may anneal to sequences other than your intended target, leading to nonspecific PCR amplification.  If the Ta is too high, you may see a reduction in reaction efficiency, as the likelihood of primer annealing is reduced.
    • GC content should be between 35-65% with an ideal content of 50%. Primer sequences should not contain regions of 4 or more consecutive G residues.
  • For more information on primer design or probe and primer design, visit IDT Designing PCR primers and probes webpage.