SPADA Draft Documents

474

Multiplexing

Multiplexing involves performing numerous assays in the same reaction chamber. 475 Multiplexing has the advantages of reducing the number of tests, thereby saving reagents, time, 476 money, and also limiting the amount of sample needed. Such multiplexing can be as small as 2- 477 plexes where the desired target is PCR amplified in the presence of an internal positive control 478 (e.g., M13 bacteriophage or RNase P control) to much larger multiplexes where numerous 479 pathogens are detected in the same reaction. The major challenge of multiplexing is to find sets 480 of primers that are “mutually compatible” under a given set of reaction conditions. “Mutually 481 compatible” means that the primer sets amplify with similar efficiency, do not cross-hybridize to 482 incorrect amplicons, do not form primer-dimers, and do not form false amplicons involving the 483 matrix background. Designing the primers to amplify at similar rates is critical to ensuring that 484 amplification of one or more targets does not overtake the reaction and consume all the reagents 485 or bind to all of the enzyme. Uniform amplification efficiency can be achieved by using the 486 principles described above (Physical Chemistry Modeling) to design primers that bind to 487 thermodynamically exposed (i.e. unfolded) regions of the target, forming amplicons that do not 488 have significant folding that can inhibit polymerase extension, and designing primers that do not 489 form competing hairpins. Experimental testing of candidate singleplexes to ensure that each one 490 amplifies efficiently and does not give a false positive in the no-template control reaction is 491 highly recommended before proceeding to multiplex testing. Minimizing the formation of 492 primer-dimers is relatively easy to check computationally (19). However, the exponential 493 explosion in the number of possible multiplex reactions makes it computationally intractable to 494 use a brute-force approach to check all possible multiplex permutations for all possible artifacts 495 that can occur (see below). 496

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