SPADA Meeting Book

conservation to near-neighbor organisms or to contaminating organisms that could cause false 369 positives. Thus, we recommend the use of k-mer algorithms to analyze inclusivity and 370 exclusivity genome databases to determine optimal locations of signature design regions. One 371 such algorithm is described in the literature by Yuriy Fofanov’s group (8) and applied to the 372 development of an assay for the 2001 pandemic H1N1 influenza A. Such a k-mer algorithm is 373 also available in the commercial PanelPlex-Consensus program (DNA Software, Inc., Ann 374 Arbor, MI). Other alternative approaches include: Uniquemer (11); BioVelocity (9); or Core/pan 375 genome analyses to identify unique genes that can be assay targets (12, 13). In all these 376 approaches, the key first step is to create the inclusivity, exclusivity and environmental 377 background panels. 381 programs (such as those available from many commercial oligonucleotide synthesis vendors) 382 utilize nearest-neighbor thermodynamic rules to compute the 2-state  G  T ,  H  ,  S  , and 383 melting temperature, Tm (15). In performing such hybridization predictions, most programs rely 384 on the 2-state melting temperature to determine hybridization quality. The Tm is intuitively 385 useful because it is the temperature at which 50 % of the target is bound by the oligonucleotide 386 and 50 % is unbound. However, the Tm does not indicate the amount of hybridization at the 387 desired annealing temperature for primers or at the extension temperature for TaqMan probes. A 388 common misconception is that the best way to design primers is to match their Tm’s (14). This 389 procedure is suboptimal, however, for two reasons: 1) even if the Tm’s are matched, the binding 390 curves have different slopes (due to different  H  values) and thus different amounts bound at 391 378 379 4.3.5 Physical Chemistry Modeling 380 Predicting the strength of primer hybridization is critical for assay design (14). Most design

Commented [LN(2]: These probably need to be defined.  Commented [SS3R2]: Will check with STL

21