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4.3.10 Multiplexing

Multiplexing involves performing numerous assays in the same reaction chamber. 492 Multiplexing has the advantages of reducing the number of tests, thereby saving reagents, time, 493 money, and also limiting the amount of sample needed. Such multiplexing can be as small as 2- 494 plexes where the desired target is PCR amplified in the presence of an internal positive control 495 (e.g., M13 bacteriophage or RNase P control) to much larger multiplexes where numerous 496 pathogens are detected in the same reaction. The major challenge of multiplexing is to find sets 497 of primers and probes that are “mutually compatible” under a given set of reaction conditions. 498 “Mutually compatible” means that the primer sets amplify with similar efficiency, do not cross- 499 hybridize to incorrect amplicons, do not form primer-dimers, and do not form false amplicons 500 involving the matrix background. Designing the primers to amplify at similar rates is critical to 501 ensuring that amplification of one or more targets does not overtake the reaction and consume all 502 the reagents or bind to all of the enzyme. Uniform amplification efficiency can be achieved using 503 the principles described above (Physical Chemistry Modeling) to design primers that bind to 504 thermodynamically exposed (i.e. unfolded) regions of the target. These designs should result in 505 amplicons that do not have significant folding that can inhibit polymerase extension and primers 506 that do not form competing hairpins. Experimental testing of candidate singleplexes to ensure 507 that each one amplifies efficiently and does not give a false positive in the no-template control 508 reaction is highly recommended before proceeding to multiplex testing. Minimizing the 509 formation of primer-dimers is relatively easy to check computationally (19). However, the 510 exponential explosion in the number of possible multiplex reactions makes it computationally 511 intractable to use a brute-force approach to check all possible multiplex permutations for all 512 possible artifacts that can occur (see below). 513

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