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The web-based multiplex PCR primer design software Ultiplex and the associated experimental workflow: up to 100- plex

Jie Yuan1,2, Ji Yi3, Meixiao Zhan4

  • 1General Surgery Center, Zhujiang Hospital, Southern Medical University, 253 Industrial Boulevard, Guangzhou, 510280, Guangdong Province, China.

BMC Genomics
|November 19, 2021
PubMed
Summary

Ultiplex is a new web-based tool for designing multiplex PCR primers, simplifying the process for complex genomic variant analysis. It successfully designed 99.7% of target primers and clustered 271 targets into a single PCR group.

Keywords:
MultiplexPrimer designSoftwareVariantWeb interface

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Multiplex PCR is crucial for analyzing genetic variants in applications like disease testing and diagnostics.
  • Designing high-multiplicity multiplex PCR primers (over 100 plex) is challenging due to potential secondary structures and nonspecific amplification.
  • Existing primer design methods require significant manual effort and experimental validation.

Purpose of the Study:

  • To develop a user-friendly, automated, and highly customizable web-based software for multiplex PCR primer design.
  • To address the need for efficient primer design in high-throughput genomic variant analysis.

Main Methods:

  • Development of Ultiplex, a free online multiplex primer design tool with a web-based interface.
  • Evaluation of Ultiplex performance by assessing primer design success rate and clustering capability.
  • Experimental validation of designed primers for detecting low-frequency mutations.

Main Results:

  • Ultiplex successfully designed 99.7% (294/295) of target primers.
  • 271 targets were clustered into a single compatible PCR group using 108 primers.
  • The designed primer group accurately detected a mutation at a 0.25% mutation rate.

Conclusions:

  • Ultiplex is an effective web-based tool for multiplex PCR primer design, offering batch design and compatibility checking.
  • The software provides flexible user-defined parameters for primer and product specifications.
  • Ultiplex simplifies genomic variant analysis and supports biological research by minimizing primer design and experimental work.