Overview over different assay development strategies.

A) After choosing the method for calling genotypes (Figure 2) 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/Tm, 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 PAH 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 HBB gene (reference). 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.