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4.3.6 Checking for Specificity and Coverage

Traditionally, the BLAST algorithm (22) is used to scan primer candidates against a database 414 of genomes to determine if the primer hybridization is specific. BLAST was developed to deduce 415 sequence similarity using evolutionary scoring, and BLAST is outstanding for such applications. 416 However, for primer design, sequence similarity is not actually the metric that matters most. 417 Instead, the quality of the complementarity to a primer is the scoring criteria that matters for 418 primer design. A better approach is to use thermodynamic scoring (i.e. hybridization  G  T or the 419 amount bound from the multi-state coupled equilibrium model). Such thermodynamic scoring 420 properly accounts for sequence and length as well as the effects of strand concentrations, salt 421 conditions and temperature. Examples of programs that perform scanning of oligonucleotides 422 against genome databases are: ThermoBLAST (14), Primer-BLAST (22), and Thermonucleotide 423 BLAST (23). A significant advantage of these programs over BLAST is their ability to not only 424 find thermodynamically stable hits, but also to evaluate if the hits are extensible by a polymerase 425 (i.e. matched pairing at the 3’-ends of the primers) and determine if pairs of primers are pointing 426 in opposite directions and within some length window (e.g., less than 1000 nucleotides) so that 427 all possible amplicons are detected (e.g., ThermoBLAST ). Notably, the various programs are not 428 all equally proficient at detecting all amplicons (e.g., some programs such as Primer-BLAST do 429 not detect mismatched hybridization very well).

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4.3.7 Probe Design

Most instrumentation for detecting a PCR reaction requires the use of a fluorescent moiety. 433 Addition of intercalating dyes such as SYBR Green (and many others) is useful for testing the 434 quality of primers for formation of a proper amplification curve (i.e. a single transition with an 435

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