SPADA Draft Documents

quality of primers for formation of a proper amplification curve (i.e. a single transition with an 409 “S” shaped saturation curve, appropriate Cq value, and curve amplitude) in the presence of target 410 genomic DNA and performing no-template controls. However, such intercalating dyes detect all 411 amplification products (i.e. both the desired amplicon and off-target amplicons) and thus dye- 412 based methods are notorious for false positives. Therefore, the use of dye-based detection is not 413 recommended for diagnostic assays. Further confirmation that the observed amplicon is the bona 414 fide target of interest requires independent amplicon sequencing (e.g,. the Sanger sequencing 415 method). For diagnostic assays, the use of an oligonucleotide probe (e.g., TaqMan, molecular 416 beacon, or capture probe) provides an extra level of specificity in that only amplicons that bind 417 to the probe are detected (and most such probe-binding amplicons are indeed the desired target 418 sequence). Comparison of the dye-based detection with the oligonucleotide probe-based 419 detection can provide invaluable confirmation that an assay is performing correctly. The 420 thermodynamic design principles for oligonucleotide probes has been review previously (14) and 421 will not be covered further here. Modified probes such as minor groove binders (MGB) and 422 locked nucleic acids (LNA) bind more tightly and specifically to their intended targets so that 423 shorter probe sequences can be used compared to probes that use only natural nucleotides. Use of 424 shorter modified probes can be particularly helpful for highly variable viruses and bacteria that 425 don’t have a large signature region available. However, a drawback of such MGB and LNA 426 probes is that they can fail to bind to new variants of the target that contain mismatches thereby 427 making such modified probes more prone to signature erosion. If one intends to use modified 428 probes, then acquiring a comprehensive inclusivity database that captures the breadth of 429 variation is an essential pre-requisite.

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