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Multiplexing primer pairs
| 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.
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| 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. |
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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).
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| 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. |
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| 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. |
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