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Primer Design for SYBR® Green Assays

  • Design Primers: Designs primers optimized for SYBR® Green assays.

  • Avoid Homology: AlleleID® carries out highly specific SYBR® Green primer design by BLAST searching your sequences, automatically interpreting the results and then designing highly specific SYBR® Green primer pairs.

  • BLAST: SYBR® Green primers can be BLASTed against genomic databases available at NCBI. This helps verify the SYBR® Green primer design and visualize primer and amplicon specificity.

  • Template Secondary Structures: Template structures identified are avoided while designing SYBR® Green primers.

  • Algorithm: Calculates SYBR® Green primer Tm using nearest neighbor thermodynamic algorithm.

  • SYBR® Green Primer Rating: Ranks the designed SYBR® Green primers according to expected priming efficiency.

  • Comprehensive Criteria: Screens SYBR® Green primers for their thermodynamic properties, location and secondary structures.

  • Multiplex Primers: Avoids cross homologies with all other dimers to reduce primer dimer.

  • Pre-designed Primers: Designs optimal TaqMan® probes and molecular beacons and for use with well proven SYBR® Green primers. Additionally, you can use a pre-designed or published or well proven forward or reverse primer and have AlleleID® design the rest of the assay. For example, for a given forward primer, AlleleID® would design the reverse primer for SYB®R Green assays and would design both the reverse primer and the TaqMan® probe for TaqMan® assays.

  • SNP Amplification: Designs SYBR® Green primers to amplify SNP sites. AlleleID® can even analyze SNP specific primers to help you decide their suitability for the reaction.

Species Identification Assays/Cross Species Assays/Allele Identification Assays

AlleleID® aligns sequences using ClustalW and analyzes conserved and species specific regions. You can then use the program for real time PCR primer design (SYBR® Green primer design included) and dual labeled probe design (TaqMan® probes, TaqMan® MGB probes and molecular beacons). These assays are designed to detect only the strain (strain detection) or species of interest from the mix.

Sophisticated Algorithms for Assay Success

Highly specific oligos are designed by avoiding regions of significant homologies found by automatically interpreting BLAST search results. Real time PCR primer & probe efficiency is enhanced by avoiding template secondary structures. "Minimal Set", one of the most innovative features in the program, helps design the fewest number of allele specific oligonucleotide primers and dual labeled probes that uniquely identify each of the desired species/strain/taxa from the mix, lowering assay costs. For taxa or cross species assays, this feature is especially useful when the group or taxa is highly dissimilar. For a partial set of pre-designed, proven set of primers, AlleleID® can design compatible primers and probes for the rest of sequences for species identification or taxa specific assays.


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