Microwell Array Based Universal Multiplex PCR And Its Application

Multiplex PCR enables to simultaneously detect and analyze multiple nucleic acid fragments in a single tube using more than one pair of primers. As a method with high efficiency, it has been widely applied on both basic biology and clinical diagnostics. However, due to the interference and competition among the primer pairs, conventional multiplex PCR is difficult to evenly and effectively amplify all the targeted fragments, especially for high throughput amplification. Conventional methods may success in ten or less than ten-plex PCR, but the optimization largely depends on the experience of the operator, and the whole process is time and labor consuming and difficult to standardize, so it is not a universal method. To address the challenge, we developed a universal multiplex PCR and subsequent detection strategy based on microwell chip combined with DNA microarray. Owing to the hydrophobic/hydrophilic surface, we constructed multiple physically isolated micro-reactions on a chip, each reaction containing one pair of specific primers and common templates, so as to completely avoid the interference and competition among the primer pairs. We examined the properties of the method by 11-plex or 116-plex PCR. Flexibility test suggests that the approach can evenly amplify and detect any combination of targets without any particular optimization; the detection limit is less 5 copies per well, even single molecule; RNA or mixture of RNA and DNA can be well amplified and detected by performing RT-PCR at the same time; in high throughput multiplex assay of single-targets, as high as 99.1% (115/116) targets obtained clear and specific bands or spots; in 116-plex PCR with all the templates, positive rate of detection is about 97.4% (112/115), suggesting its potential for high throughput detection. We also analyzed the deletion of exons of DMD gene from 10 clinical samples using our strategy, and all but one perfectly matched with the clinical records. Our results fully demonstrate that the microwell chip based multiplex PCR is a universal and powerful method for rapid, effective and convenient detection multiple targeted genes or species simultaneously, and has a great potential for practical application. Lastly, we summarized the protocol to perform multiplex PCR and detection of products based on the experiments.