( other additional larger PCR products ...)QIAGEN Multiplex PCR Handbook

other, additional larger PCR products may be amplified from these outside primers (Figure 2).

Generation of Additional PCR Products in Exon-Specific PCR



Figure 2. Two exons from the human HFE gene were amplified from human genomic DNA either separately, (Exon 4); (Exon 2), or together (Exon 2 and 4). The PCR products generated from Exon 4 and Exon 2 were 395 bp and 242 bp respectively. However, in the multiplex PCR an additional product of 2219 bp is observed, generated by the 2 outside primers.

8. Optimization of reaction conditions for special multiplex PCR applications

The QIAGEN Multiplex PCR Kit protocols have been developed to give satisfactory results in most cases. In some special cases, modifications to the conditions given in the protocol may improve performance.

Large number of PCR products: For multiplex PCR reactions with more than 10 PCR products, a decrease of primer concentration to 0.1 M may lead to a more uniform product yield. The annealing time can also be increased from 90 seconds to 3 minutes. To establish a multiplex system with a large number of PCR products, it is strongly recommended to check the primer concentration given by the primer supplier. The use of high-quality primers, for example HPLC purified, is recommended, although standard quality may also be sufficient. We strongly recommend only combining primers of comparable quality. Highly sensitive applications or
low template amount: Increasing the annealing time from 90 sec
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