SPADA Draft Documents

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Checking for Specificity and Coverage

Traditionally, the BLAST algorithm (22) is used to scan primer candidates against a database 388 of genomes to determine if the primer hybridization is specific. BLAST was developed to deduce 389 sequence similarity using evolutionary scoring, and BLAST is outstanding for such applications. 390 However, for primer design, sequence similarity is not actually the metric that matters most. 391 Instead, the quality of the complementarity to a primer is the scoring criteria that matters for 392 primer design. A better approach is to use thermodynamic scoring (i.e. hybridization ∆ G ° T or the 393 amount bound from the multi-state coupled equilibrium model). Such thermodynamic scoring 394 properly accounts for sequence and length as well as the effects of strand concentrations, salt 395 conditions and temperature. Examples of programs that perform scanning of oligonculeotides 396 against genome databases are: ThermoBLAST (14), Primer-BLAST (22), and Thermonucleotide 397 BLAST (23). A significant advantage of these programs over BLAST is their ability to not only 398 find thermodynamically stable hits, but also to evaluate if the hits are extensible by a polymerase 399 (i.e. matched pairing at the 3’-ends of the primers) as well as to determine if pairs of primers are 400 pointing in opposite directions and within some length window (e.g., less than 1000 nucleotides) 401 so that all possible amplicons are detected (e.g., ThermoBLAST ). Notably, the various programs 402 are not all equally proficient at detecting all of the amplicons (e.g., some programs such as 403 Primer-BLAST do not detect mismatched hybridization very well).

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Probe Design

Most instrumentation for detecting a PCR reaction requires the use of a fluorescent moiety. 407 Addition of intercalating dyes such as SYBR Green (and many others) is useful for testing the 408

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