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BLAST (23). A significant advantage of these programs over BLAST is their ability to not only 415 find thermodynamically stable hits, but also to evaluate if the hits are extensible by a polymerase 416 (i.e. matched pairing at the 3’-ends of the primers) and determine if pairs of primers are pointing 417 in opposite directions and within some length window (e.g., less than 1000 nucleotides) so that 418 all possible amplicons are detected (e.g., ThermoBLAST ). Notably, the various programs are not 419 all equally proficient at detecting all amplicons (e.g., some programs such as Primer-BLAST do 420 not detect mismatched hybridization very well). 424 Addition of intercalating dyes such as SYBR Green (and many others) is useful for testing the 425 quality of primers for formation of a proper amplification curve (i.e. a single transition with an 426 “S” shaped saturation curve, appropriate Cq value, and curve amplitude) in the presence of target 427 genomic DNA and performing no-template controls. However, such intercalating dyes detect all 428 amplification products (i.e. both the desired amplicon and off-target amplicons) and thus dye- 429 based methods are notorious for false positives. Therefore, the use of dye-based detection is not 430 recommended for diagnostic assays. Further confirmation that the observed amplicon is the bona 431 fide target of interest requires independent amplicon sequencing (e.g., the Sanger sequencing 432 method). For diagnostic assays, the use of an oligonucleotide probe (e.g., TaqMan, molecular 433 beacon, or capture probe) provides an extra level of specificity in that only amplicons that bind 434 to the probe are detected (and most such probe-binding amplicons are indeed the desired target 435 sequence). Comparison of the dye-based detection with the oligonucleotide probe-based 436 detection can provide invaluable confirmation that an assay is performing correctly. The 437 421 422 4.3.7 Probe Design 423 Most instrumentation for detecting a PCR reaction requires the use of a fluorescent moiety.

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