Designing primers and probes for quantitative (also known as real-time) PCR (qPCR) is not an easy task because both the primer and probe sequences need to be considered during design. For me, Beacon Designer is the best way to design qPCR primers and probes. This software allows us to design Molecular Beacons probes for standard qPCR and for Nucleic Acid Sequence Based Amplification (NASBA), TaqMan® probes for standard qPCR and as well as locked nucleic acid (LNA)- substituted TaqMan® probes, other FRET (fluorescent resonance energy transfer) probes, and SYBR® Green primers for real-time detection of PCR products.
The software is fully loaded with a number of features which make it the best option for primer and probe design. For example, for Molecular Beacons probes, Beacon Designer has an automatic Tm adjustment in which it automatically selects the optimal length such that the probe does not dimerize or form hairpin structures. The Tm calculation is highly accurate; it is calculated by connecting to the mfold server. The software also gives us a graphical view of the Beacons probe. The software can also design and optimize primers and probes for TaqMan® assays. Just like Molecular Beacon, TaqMan® probes are free of repeats and do not dimerize. Beacon Designer also allows for the design of multiplex assays. The Tm calculation for TaqMan® probes is based upon the SantaLuia values; Tm calculations utilize “nearest neighbor” thermodynamic values. A TaqMan® probe graphical view is also available. All these feature are also available with FRET probes and primers for SYBR® Green detection.
In addition to probe design, Beacon Designer can also design the forward and reverse primers for Molecular Beacons, TaqMan®, FRET and SYBR® Green qPCR experiments. The Tm calculation for the primer pair is also based on the Santa Lucia values. In addition, the software also gives a rating to the primers based upon their priming efficiency. Primers are also screened for there thermodynamic properties, for secondary structures, and cross homologies (i.e. to reduce primer dimmers). The main feature I like about the software is that we can also use previously designed primers with chemistries different from what they were originally designed for. For example, primers used in SYBR® Green detection can also be used in a TaqMan® assay.
We can also BLAST the primer and probe sequences through the Beacon Designer software. The software opens a separate window for the BLAST results; results include the any sequence hits for the primer-pair, amplicon and probe. Specific sequences can be retrieved using accession numbers.
The software can also generate reports in which it mentions important points characteristics such as Tm, delta G, product size, sequence and as well as providing a ranking of the predicted performance of the primers and probes.
If we are designing primers and probes for a known sequence, this can be done in just a few clicks. By entering a stretch of sequence or the gene’s accession number, the software can directly access and upload the sequence from Entrez. We can even then edit the sequence. The software also allows hundreds of SNPs to be loaded for designing probes for multiplex detection
To give a summary of using the Beacon Designer software: First, select the desired sequence and the type of probe you wish to design (i.e. SYBR®, TaqMan®, etc.). Then perform a BLAST search of the desired sequence followed by a template structure search which avoids secondary structure while designing primers. After performing these two searches, just click onto the Evaluate button for your primer and probe designs. As per the requirement of the qPCR assay, we can vary different conditions like length and Tm for the amplicon, both primers as well as for the probe. We can also set the advanced parameters like hairpin maximum delta-G, self-dimer maximum delta-G, etc.
We have used this software mainly to design primers and probes for TaqMan® and for SYBR® Green assays. The qPCR reactions are working very efficiently.
A demo version of Beacon Designer is freely available from Premier Biosoft (http://www.premierbiosoft.com/molecular_beacons/index.html) You all must try it at once. I only wish this software could design Scorpions® probes also.
As a whole, Beacon Designer is a useful and efficient tool for designing sequences for qPCR in just few clicks.
Product Specialist
Molecular Diagnostics
Biotron