Overview over different assay development strategies.

<p><b>A</b>) After choosing the method for calling genotypes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014777#pone-0014777-g002" target="_blank">Figure 2</a>) or for genotyping loci, placed in genomic regions with wide to narrow ranges in G+C content, the choice to run the assay at a single or at many assay stringencies is made. Based on the above selections “Input criteria” the parameters (Opt. Param.) that need to be optimized/flexible are shown below with the achieved success rate (SR) (percentage of mutations successfully genotyped). The parameters that must be optimized are probe length/<i>T</i>m, spacer length (position of probe relative to array surface) and alternative combinations of wildtype and mutant probe in probe-pairs (Alt probes). The success rate obtained in this study (genotyping of <i>PAH</i> mutations) is valid for the wide range in G+C content. The results from a narrow range in G+C content are from genotyping mutations in the <i>HBB</i> gene (reference). <b>B</b>) The steps in bottom-up (left) and top-down (right) assay strategies are listed. The bottom-up approach is an iterative process with many rounds of probe design, testing and the redesigning of probes. In contrast the top-down approach only utilizes one optimization experiment including all parameters needed for a functional assay.</p>

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