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Non-specific Detection using DNA Binding Dyes
In real time PCR, DNA binding dyes are used as fluorescent reporters to monitor the real time PCR reaction. The fluorescence of the reporter dye increases as the product accumulates with each successive cycle of amplification. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase. If a graph is drawn between the log of the starting amount of template and the corresponding increase the fluorescence of the reporter dye fluorescence during real time PCR, a linear relationship is observed.
a. Molecular Beacons
b. TaqMan® Probes
c. FRET Hybridization Probes
d. Scorpion® Primers
AlleleID® designs exon probes. Such probes are important to confirm the presence of an exon in the variant.
Technical resource on Gene Splicing.
Taxa Specific/Cross Species Identification: Probes with Mismatched Bases and Conserved Primer
Probes with Mismatched Bases and Conserved/Optimal Primers: AlleleID® can design probes with specified number of mismatch bases. The mismatched bases will follow the majority consensus, meaning that the probe will have the most common base within the alignment. This design minimizes cost, an important consideration, especially in diagnostic assay design. The primers are designed in the conserved regions. To reduce assay costs, it designs the minimal number of primer pairs that are required to amplify the conserved regions across the alignment.
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