( y increasing the annealing time from ...)QIAGEN Multiplex PCR Handbook

y increasing the annealing time from 90 seconds to 3 minutes. However, some alleles may not be detectable when using low amounts of template DNA, due to the fact that not all target loci are present in the reaction. This is caused by stochastic fluctuation.

7. Guidelines for special multiplex PCR applications

Transgene detection
Genetically modified animals and plants can be generated to study the function of particular genes. Targeted mutations can be introduced that alter the function of the gene locus of interest, for example by inactivating or modifying its function. This allows research on the role of certain genes in complex biological processes. Modified genomic loci can be easily distinguished by multiplex PCR. To distinguish the wild-type from the mutant gene locus, pairs of primers should be designed that are specific either for the wild-type locus or for the mutant locus. In Figure 1, one primer is located on the wild-type locus. Two reverse primers have been designed, so that one primer is specific for the wild-type locus and the other specific for the mutant locus, resulting in PCR products of different sizes. Multiplex PCR is highly suited for the efficient detection of transgenes. In this case, two primer pairs can be used: one pair specific for the introduced transgene and the other pair specific for a wild-type DNA sequence. This second primer pair acts as a control for the amount and quality of the template DNA.

Detection of Transgenes


Figure 1. Transgenic mice were screened using the QIAGEN Multiplex PCR Kit and a set of 3 primers specific for the recombination activating gene 2 locus. The primers were designed to
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