Fig. 9. Single-locus PCR with 34 different primer pairs using the same cycling conditions. Arrows indicate position of the specific products in lanes 25, 28 and 33, in which other unspecific products also appear. Such primer pairs are difficult to use both by themself and in multiplex PCR. However, even though some unspecific products still appeared, primer pair 28 was multiplexed in mixture 5 (Figure 1) and used in a microdeletion screening project. The unspecific products did not interfere with data interpretation. Examples of multiplex reactions using these primers are shown in Fig. 1.

Fig. 7 (duplicate). Single locus PCR and multiplex PCR with equimolar amounts of primers from mixture K, performed in the same cycling conditions. In Some products of mixture K become weak or invisible, requiring further adjustment of primer amount(s) and of cycling conditions. Primers used in mixture K amplify polymorphic loci, explaining the appearence of multiple bands on a nondenaturing agarose gel.

Fig. 10. Equimolar amounts of the same primers used for mixture K (see also Fig. 7 above), where amplified in pairs. In lanes 1, 2 and 4, one locus was amplified less efficiently than the other one (arrows). As mentioned before, amplification of the "weaker" loci can be improved increasing the amount of primers or adjusting the reaction conditions.