SPADA Docs

“S” shaped saturation curve, appropriate Cq value, and curve amplitude) in the presence of target 436 genomic DNA and performing no-template controls. However, such intercalating dyes detect all 437 amplification products (i.e. both the desired amplicon and off-target amplicons) and thus dye- 438 based methods are notorious for false positives. Therefore, the use of dye-based detection is not 439 recommended for diagnostic assays. Further confirmation that the observed amplicon is the bona 440 fide target of interest requires independent amplicon sequencing (e.g., the Sanger sequencing 441 method). For diagnostic assays, the use of an oligonucleotide probe (e.g., TaqMan, molecular 442 beacon, or capture probe) provides an extra level of specificity in that only amplicons that bind 443 to the probe are detected (and most such probe-binding amplicons are indeed the desired target 444 sequence). Comparison of the dye-based detection with the oligonucleotide probe-based 445 detection can provide invaluable confirmation that an assay is performing correctly. The 446 thermodynamic design principles for oligonucleotide probes have been reviewed previously (14) 447 and will not be covered further here. Modified probes such as minor groove binders (MGB) and 448 locked nucleic acids (LNA) bind more tightly and specifically to their intended targets so that 449 shorter probe sequences can be used compared to probes that contain only natural nucleotides. 450 Shorter modified probes can be particularly helpful for highly variable viruses and bacteria that 451 do not have a large signature region available. However, a drawback of such MGB and LNA 452 probes is that they can fail to bind to new variants of the target that contain mismatches, thereby 453 making such modified probes more prone to signature erosion. If one intends to use modified 454 probes, then acquiring a comprehensive inclusivity database that captures the breadth of 455 variation is an essential pre-requisite.

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