SPADA Draft Documents

363

Physical Chemistry Modeling

Predicting the strength of primer hybridization is critical for primer design (14). Most design 364 programs (such as those available from many commercial oligonucleotide synthesis vendors) 365 utilize nearest-neighbor thermodynamic rules to compute the 2-state ∆ G ° T , ∆ H ° , ∆ S ° , and 366 melting temperature, Tm (15). In performing such hybridization predictions, most programs rely 367 on the 2-state melting temperature to determine hybridization quality. The Tm is intuitively 368 useful because it is the temperature at which 50% of the target is bound by the oligonucleotide 369 and 50% is unbound. However, the Tm does not indicate the amount of hybridization at the 370 desired annealing temperature for primers or at the extension temperature for TaqMan probes. A 371 common misconception is that the best way to design primers is to match their Tm’s (14). This 372 procedure is suboptimal, however, for two reasons: 1) even if the Tm’s are matched, the binding 373 curves have different slopes (due to different ∆ H ° values) and thus different amounts bound at 374 the annealing temperature; and 2) the 2-state Tm does not capture the competing unimolecular 375 secondary structures. Primer and target unimolecular secondary structure can be predicted using 376 dynamic programming algorithms such as MFOLD (16), RNAStructure (17) or OMP (14). 377 Rather than focusing on Tm-based metrics, it is recommended to use software that focuses on 378 solving the competing equilibrium for the actual amount bound at the desired temperature. 379 Computation of the amount bound is best accomplished using a multi-state coupled equilibrium 380 model (14, 18). In addition to computing bimolecular hybridization and competing unimolecular 381 folding, it is useful to check sets of primers to ensure that they do not form primer-dimer species 382 involving the 3’-ends of the primers. This can be predicted with programs such as AutoDimer 383 (19) and ThermoBLAST (14). There are also a variety of experimental approaches for eliminating 384 primer-dimers (20, 21). 385

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