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Single locus PCR.First step in designing a multiplex PCR is choosing the primer pairs which can be combined. One important requirement is to find a PCR program allowing optimal amplification of all loci when taken individually (Fig. 9). This is achieved by adjusting the annealing and extension time and temperature.
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.
Multiplexing equimolar primer mixtures.The next step is combining the desired primer pairs in multiplex mixture(s), using equimolar amounts of each primer. PCR amplification of the multiplex mixtures can be performed, first using exactly the same PCR program as with individual primer pairs. Very often, this will results in preferential amplification of some loci. Such a situation will require further adjustment in cycling conditions and primer concentration. Although, sometimes unspecific products can be seen in single-locus PCR (yellow arrow in PCR product # 2), these unspecific products usually become invisible when the multiplex reaction is performed. This is probably due to the concurrent ampification of many specific loci, which overwhelms the unspecific products (although they are probably still present in small quantities).
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.
Adjustment of cycling conditions
For example, figure 11 illustrates the influence of the extension temperature. Equimolar primer mixtures A-D were amplified using two different PCR programs, one at 65o C (yellow lanes) and the other at 72o C (green lanes) extension temperature. In general, there is a higher yield of PCR products for A, B and D when program A was used. This shows that the 72o C extension temperature, negtively influenced amplification of some loci (pink arrows),while also making some unspecific products visible (yellow arrows). It is likely that, for the short PCR products used in these examples (below 500 bp), the higher annealing temperature is probably detrimental to the stability of the DNA helix, so less strands of DNA have the chance to become "copied" by the polymerase after annealing.
Fig. 11. Example of the influence of extension temperature. Multiplex PCR with mixtrues A-B using two different PCR programs. Reactions on the right side (green) were performed in identical cycling conditions with Fig. 9, whereas reactions on the left side (yellow) were performed using cycling conditions in which extension temperature was dropped from 72 o C to 65 o C. Reaction worked more efficiently with the lower extension temperature (pink arrow show missing products, yellow arros show unspecific products).
Primer amount and buffer concentration.To improve the amplification of some of the DNA products from Fig. 11 above, the amount of primers was increased 2-5x for those loci. At the same time, the PCR buffer concentration was increased to 2x. These modifications allowed a much more efficient and reproducible amplification, with no unspecific products.
Fig. 12.Multiplex PCR with mixtures A-D, in cycling conditions similar to the ones on the left side of Fig. 11 above (annealing at 65 o C), but using 2x PCR buffer. The amount of primer pairs was increased for some of the weak products from Fig. 11. Cleaner and more efficient amplifications were obtained.