Multiplex primer design

Multiplex PCR is an approach commonly used to amplify several DNA target regions in a single PCR reaction and it is great technology for an endless list of applications. The greatest potential for multiplex PCR is the acceleration of the number of reactions that can be carried out simultaneously. Further improvement can be achieved by selecting the optimal set of primers that maximize the range of common Tm.

FastPCR quickly calculates multiplex PCR primer pairs for given target sequences or different targets inside sequence or both ways. The speed of calculation depends from amount of target sequence and amount primer pair for each of them. Other way for design compatible multiplex PCR primer pairs is use the predesigned as references for new designed primers.

The user can also input options for the PCR product involving the minimum product size differences among the set of designed primer pairs. It also allows to set primer design conditions individually for each given sequences or using common options. The individual setting have highest priority to PCR primer or probe design than general settings. The result includes primer sequences for individual sequences, their compatible primer pairs with product size and annealing temperature and final result for compatible primer pairs for each sequence with all information includes primer pair sequences, product size and annealing temperature. It is ideally to design all primer pairs with near equal annealing temperature in single reaction. For most cases the multiplex PCR conditions are resisting to a small variation (up 3°C) of Ta between all primer pairs and PCR products. Designing primers with the same dG will render more efficient primers pairs, matching Tm’s is a less accurate approach than matching dG’s. Synchronizing Tm for primer pair user can control from “Primer Design Options” or with command: -ptms3.

The annealing temperature must be optimal in order to guarantee effective amplification of the targets genomic sequence while minimizing the risk of unspecific amplification. To amplify the target genomic sequence effectively, the primers “quality” and properties should be highest. PCR primer design for multiplex PCR can be performed for standard or inverted PCR pairs or both of them. A minimum of two sequences (or two targets in single sequence) must be implemented for this analysis. The program will find the compatible primer pairs for each sequence and will make a continuous numbering of pairs for all investigated sequences.

Another feature of the program, user can select not only compatible pairs of primers, as well as compatible single primers for different targets or sequences. That is, program can design both pairs of primers and single primers or only single primers for different targets or sequences: