SPADA Meeting Book

thermodynamic design principles for oligonucleotide probes have been reviewed previously (14) 438 and will not be covered further here. Modified probes such as minor groove binders (MGB) and 439 locked nucleic acids (LNA) bind more tightly and specifically to their intended targets so that 440 shorter probe sequences can be used compared to probes that contain only natural nucleotides. 441 Shorter modified probes can be particularly helpful for highly variable viruses and bacteria that 442 do not have a large signature region available. However, a drawback of such MGB and LNA 443 probes is that they can fail to bind to new variants of the target that contain mismatches, thereby 444 making such modified probes more prone to signature erosion. If one intends to use modified 445 probes, then acquiring a comprehensive inclusivity database that captures the breadth of 446 variation is an essential pre-requisite. 450 scoring equation. Unfortunately, there is no agreed upon “currency” for goodness of primer 451 performance. Instead there are many metrics that have vastly different units, such as free-energy 452 differences for folding and hybridization, amount bound, amplicon folding, target conservation, 453 off-target hybridization, primer dimerization, primer-amplicon cross-hybridization, and a long 454 list of non-thermodynamic rules (G-quartets, sequence complexity (or information entropy), 455 amplicon length, etc.). It is still something of an art to combine all of these disparate scoring 456 terms into one big equation and produce a result meaningful to a user (such as a final score that 457 ranges from 1 to 100). In addition, there is no agreement in the community as to what the relative 458 weighting of different scoring terms should be. For this reason, it is recommended to use 459 software that exposes the scoring equation and the weights used for each scoring term (e.g., 460 447 448 4.3.8 Ranking the Design Results 449 A critical part of primer design is the ranking of the candidate designs using some sort of

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