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AlleleID® Provides a Number of Options for Species Identification
Unique Probes with Unique Primers: When the probe type chosen is Unique, AlleleID® designs probes to identify each sequence in the Bind To box, one probe per sequence. The program searches for base positions that are unique to each individual sequence in the alignment. The probes are centered as close as possible over these unique bases, so that even when the mismatch is only one base, the assay succeeds.
Limitations of End-point PCR
In a PCR reaction as the reaction progresses, the reagents are being consumed as a result of amplification. Now the PCR product is no longer being doubled at each cycle due to this reagent constraint. This depletion will occur at different rates for each replicate. Thus, the samples begin to diverge in their quantities. This diminished amplification is the linear phase of the reaction. The plateau for each tube will differ due to the different reaction kinetics for each sample. It is in this phase where traditional PCR takes its measurement, also known as the end-point. This End-Point Detection has some problems such as low resolution, poor precision, low sensitivity and the need for post PCR processing. Also, the results of this detection are not expressed in numbers and there is no scope for automation.
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.
Minimal Probes with Optimal Primers
For Optimal, the program minimizes the number of primers required to amplify all sequences. The goal is to minimize the synthesis and overall assay costs.
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