SPADA Meeting Book

the annealing temperature; and 2) the 2-state Tm does not capture the competing unimolecular 392 secondary structures. Primer and target unimolecular secondary structure can be predicted using 393 dynamic programming algorithms such as MFOLD (16), RNAStructure (17) or OMP (14). 394 Rather than focusing on Tm-based metrics, it is recommended to use software that focuses on 395 solving the competing equilibrium for the actual amount bound at the desired temperature. 396 Computation of the amount bound is best accomplished using a multi-state coupled equilibrium 397 model (14, 18). In addition to computing bimolecular hybridization and competing unimolecular 398 folding, it is useful to check sets of primers to ensure that they do not form primer-dimer species 399 involving the 3’-ends of the primers. This can be predicted with programs such as AutoDimer 400 (19) and ThermoBLAST (14). There are also a variety of experimental approaches for eliminating 401 primer-dimers (20, 21). 405 of genomes to determine if the primer hybridization is specific. BLAST was developed to deduce 406 sequence similarity using evolutionary scoring, and BLAST is outstanding for such applications. 407 However, for primer design, sequence similarity is not actually the metric that matters most. 408 Instead, the quality of the complementarity to a primer is the scoring criteria that matters for 409 primer design. A better approach is to use thermodynamic scoring (i.e. hybridization  G  T or the 410 amount bound from the multi-state coupled equilibrium model). Such thermodynamic scoring 411 properly accounts for sequence and length as well as the effects of strand concentrations, salt 412 conditions and temperature. Examples of programs that perform scanning of oligonucleotides 413 against genome databases are: ThermoBLAST (14), Primer-BLAST (22), and Thermonucleotide 414 402 403 4.3.6 Checking for Specificity and Coverage 404 Traditionally, the BLAST algorithm (22) is used to scan primer candidates against a database

22