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4.3.10 Multiplexing

Multiplexing involves performing numerous assays in the same reaction chamber. 501 Multiplexing has the advantages of reducing the number of tests, thereby saving reagents, time, 502 money, and also limiting the amount of sample needed. Such multiplexing can be as small as 2- 503 plexes where the desired target is PCR amplified in the presence of an internal positive control 504 (e.g., M13 bacteriophage or RNase P control) to much larger multiplexes where numerous 505 pathogens are detected in the same reaction. The major challenge of multiplexing is to find sets 506 of primers and probes that are “mutually compatible” under a given set of reaction conditions. 507 “Mutually compatible” means that the primer sets amplify with similar efficiency, do not cross- 508 hybridize to incorrect amplicons, do not form primer-dimers, and do not form false amplicons 509 involving the matrix background. Designing the primers to amplify at similar rates is critical to 510 ensuring that amplification of one or more targets does not overtake the reaction and consume all 511 the reagents or bind to all of the enzyme. Uniform amplification efficiency can be achieved using 512 the principles described above (Physical Chemistry Modeling) to design primers that bind to 513 thermodynamically exposed (i.e. unfolded) regions of the target. These designs should result in 514 amplicons that do not have significant folding that can inhibit polymerase extension and primers 515 that do not form competing hairpins. Experimental testing of candidate singleplexes to ensure 516 that each one amplifies efficiently and does not give a false positive in the no-template control 517 reaction is highly recommended before proceeding to multiplex testing. Minimizing the 518 formation of primer-dimers is relatively easy to check computationally (19). However, the 519 exponential explosion in the number of possible multiplex reactions makes it computationally 520 intractable to use a brute-force approach to check all possible multiplex permutations for all 521 possible artifacts that can occur (see below). 522

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