Method: New and improved multiplex PCR

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Published on: August 30, 2012

Highlighted article: Daxing Wen and Chuqing Zhang (2012) Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments. Plant Methods 8:32 (Online preview) doi: 10.1186/1746-4811-8-32


Multiplex PCR allows amplification of multiple targets in a single PCR experiment. It is possible to amplify several sections of a single template, or to amplify different templates using a number of primer sets. If there are multiple primers in a reaction, it can be difficult optimise the PCR reaction to maximise the efficiency of every primer, and it is likely that some cross-hybridisation and mis-priming will occur.

Figure 3B from Wen and Zhange (2012). A comparison of multiplex PCR (Lanes 1-4) and universal multiplex PCR (lanes 5-8), using the same primers with universal adaptors. The band intensity from traditional PCR is very variable, but it is consistently strong when the universal adaptors are used. 

Image credit: BioMed Central


The Method

Wen and Zhang from Shandong Agricultural University have devised a way around the inconveniences of multiplex PCR to develop a universal multiplex PCR method. ‘Universal adaptors’ are linked to specific primers, making the annealing temperature of the adaptor-primer structures 70°C.



The method requires a ‘Two-Step’ mode PCR programme. The first three cycles consist of several 20s annealing steps – one for every primer, so the temperatures for each step are optimised for a specific primer. These initial cycles ease the long adapter-primers into the PCR reaction. In the first two cycles, the specific primer section of the adaptor-primer binds to the template DNA. In the second cycle, the template for the Universal adapter is synthetised so that in the third cycle the full adaptor-primer sequence is amplified with the target sequence. Following the initial three long cycles are 30 ‘normal’ PCR cycles with an annealing temperature of 70°C.


Wen and Zhang used the universal multiplex PCR method to detect five simple sequence repeats in a maize seeds lot to assess the genetic purity of the batch in one step. This is a far more efficient means of testing genetic purity than assessing seed morphology or using protein and isozyme electrophoresis, which can be compromised by environmental conditions and human error.

This method does not work with common adaptor primers, but appears to be effective with all specific primers. The two-step universal multiplex PCR method could also be applied to species identification, polymorphism analysis and quantitative assays.


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