1. Innovative Algorithms

a) Statistical Optimization: The sophisticated search algorithm calculates all properties of every possible primer and probe within the allowed length and positional boundaries. Every selected primer and probe must meet all specified criteria. It is rejected if any parameter falls outside of its tolerance limits. Beacon Designer makes the task of choosing the best possible oligo set as easy as clicking a button. For expert users a list of alternate oligo sets is available and is sortable by several key parameters.

Note: Competitive products use the consecutive screening technique to choose oligos. Possible oligos are screened for each parameter in an arbitrary order for acceptable parameter values and outliers are eliminated in each screen. The result is highly dependent on the order of screening and does not result in the selecting the best oligo.

b) Avoid Cross Homologies: This function is one of the most unique features of Beacon Designer and it ensures that the oligo sets designed result in specific amplification and exact detection, BLAST search is run for the templates against the non-redundant database of the organism and the results are interpreted to identify regions of significant cross homologies automatically.

Note: We are not aware of any product that offers this functionality.

c) Default Parameter Selection: Beacon Designer provide default parameter values based on considerable research and private communications with several experts and some of the inventors of the qPCR technique. The default parameters work well for most assays. For more challenging assays, complete manual control is available.

Note: This is a quote from Alex Ryncarz, Epigenomics Inc., a long time customer of Beacon Designer-”Unless the sequence is exceptionally GC rich or repetitive, I found that I could simply use the default parameters. It is not a surprise that some sequences, especially those shorter than 500 bases and or having a high GC content, do not provide primer sets or probes with the default conditions. The inability of the software’s default conditions to provide primer or probe designs is an attribute to the consistently high quality of primer and probe designs that are produced using Beacon Designer. Other design software may also provide good results, but I have only seen excellent consistency with Beacon Designer, which saves both money and time.”

2. Many qPCR Assay Technologies

SYBR® Green, TaqMan®, MethyLight, beacons, FRET: Beacon Designer enables you to design almost all the popular qPCR technologies including SYBR® Green, TaqMan®, MethyLight, molecular beacons and FRET assays. You can even first test the primers with a SYBR® Green assay before taking the next step of purchasing the more expensive fluorescent labeled probes for any of the three dual labeled technologies.

Note: Competitive software offers only the technology the vendor recommends and expects the user to buy the reagents from them. Primer Xpress supports only TaqMan® assays and Light Cycler software supports FRET exclusively.

3. NASBA®: NASBA®, a highly sensitive and specific amplification reaction that offers several advantages over other mRNA amplification methods. NASBA® is an isothermal reaction performed at 41C, which obviates the need for a thermal cycler and may facilitate the production of point-of-test devices. A single-stranded antisense RNA product is produced during NASBA®, which can be directly hybridized by a probe sequence to accelerate post-amplification interrogation of the product. In contrast to PCR, background DNA does not interfere with the NASBA® reaction, as single-stranded RNA sequences are specifically targeted. It holds a great promise in diagnostic assay development.

Note: No other program, to our knowledge, offers NASBA® qPCR assay capability.

4. LNA: Locked Nucleic Acid or LNA probes are gaining popularity rapidly for SNP detection qPCR Assay because of improved real-time PCR assay success. Incorporation of the LNA bases significantly increases thermal stability, making shorter probes possible for standard reaction conditions.

Note: No other program, to our knowledge, offers easy to use design capability for qPCR assays with LNA substituted probes.

5. MethyLight: 5-methylcytosine is the most frequent covalent base modification of the DNA of eukaryotic cells. The development of MethyLight assay for DNA methylation analysis has significantly helped understanding biological processes such as transcription regulation, genetic imprinting, and tumorigenesis. MethyLight assay accurately determines the relative prevalence of a particular pattern of DNA methylation. The oligonucleotides designed for MethyLight assays allows high degree of specificity, sensitivity, and flexibility for methylation studies.

6. Scorpions®: Using Beacon Designer, design Scorpions® to achieve sequence-specific priming and PCR product detection using a single oligonucleotide. Scorpions® represent the most recent development in real-time PCR chemistry. The stem-loop format is a common approach for specific detection of PCR products in which the probe element and primer element are physically coupled during the reaction. It is ideally suited for many applications that require rapid detection such as viral load testing and RNA profiling.

7. Multiplex Reactions: Beacon Designer helps you design an optimum set of primers and probes for single tube multiplex Taqman® and molecular beacon assays having up to four sequences.

It first designs the best 50 or so primer and probe assays for each sequence. Then the fluorescent probe in each assay is checked for cross dimerization with the primers and probes of all the assays of the other sequences in the reaction. Potential multiplex sets are created by grouping one successful assay for each sequence in all possible combinations. Each multiplex set is then rated according to the formula described earlier, but with the addition of the various cross dimer energy and Tm mismatch terms. The resultant multiplex set rating is a function of the rating of each of the component assays and the compatibility of each assay with the other assays of the multiplex set.

With 50 or so assays for each of the four sequences, there are more than 6 million multiplex sets possible. Each multiplex set has a total of 108 cross compatibility terms in its rating. Thanks to fast computers, efficient programming, and innovative algorithms, nearly a billion interactions are resolved in a flash.

Note: Multiplexing reactions are crucial in the diagnostic assay development, which is one of the most important applications of qPCR technology. No other program, to our knowledge, has the required capability.