AlleleID® designs specific capture probes for direct hybridization assays and primers for Allele Specific PCR Extension (ASPE) assays. The assays are used for high throughput SNP applications such as genotyping, pathogen detection, strain typing, and haplotyping. To ensure specificity, the oligos are designed after avoiding the regions of homologies identified by a BLAST search.
AlleleID® then checks the oligos for cross reactivity and minimizes Tm mismatches to give you the best possible multiplex set. In the process, it analyzes millions of possible multiplex sets in an a few seconds and presents a list of alternate sets to you as well.
1. Strain Identification Assays
2. Direct Hybridization Assays
3. ASPE Assays
AlleleID® designs strain differentiation multiplex assays for Suspension Array systems based on Luminex's xMAP® technology. If you are working with closely related organisms, this functionality will enable you to run experiments in a single reaction vessel.
With a multiple sequence alignment functionality at its core, the sequences are first aligned, the species specific regions are identified and probes are designed to detect the strain of interest. The probes are screened for homologies with itself and with all other probes in the pool, assuring they work well in a multiplex reaction.
AlleleID® designs optimal allele specific capture probes for direct hybridization assays. The probes are designed such that the mutations are at their centers to maximize differentiation. Our innovative and proprietary algorithm analyzes each one of these probes for their suitability in a multiplex reaction. The probes are screened for cross and self dimers, assuring strong signal strength.
This functionality makes AlleleID® the only software product capable of designing probes for 100 individual targets in a multiplex reaction. Also included is the ability to design compatible primer pairs for amplifying the template.
A series of probes can also be designed for detecting all the variable sites of a medium to large genomic sequence. For such cases, AlleleID® designs allele specific capture probes after BLAST searching the sequences and avoiding the regions of homology.
Another approach to detect mutations is to use a solution-based sequence specific enzymatic reaction such as Allele Specific Primer Extension (ASPE) to determine the target genotype. AlleleID® designs highly specific primers for multiplexed ASPE assays. The ASPE primers are designed such that the mutations lies at their 3' end. AlleleID® also designs primers to amplify the target sequence for ASPE 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.
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.