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Modern Techniques for Species Identification
AlleleID® designs both real time PCR assays, including molecular beacons, TaqMan® probes, TaqMan® MGB, and SYBR® Green primers), and microarrays for bacterial identification, pathogen detection and taxa/species identification. AlleleID® uses the fast and accurate ClustalW algorithm to align sequences to identify the species specific regions.
Choices for Primer Design: There are two choices available for primer design, Unique and Optimal. When you choose Unique, the program designs a primer pair to amplify each sequence. 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.
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.
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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