PlexPrime™


PlexPrime™ is a novel method for nucleic acid amplification that creates amplicons which are distinctly different from the parent sequence. The combination of PlexPrime™ and PlexZyme™ technology enables multiplex mutation detection with high sensitivity and specificity.1


Features:

Multiplex compatible

  • Detect multiple mutations in a single reaction

Versatile

  • Flexibility for challenging sequences

Highly Specific

  • Increased stringency due to insert sequence

 


PlexPrimeTechFig1

Figure 1: The PlexPrimer™ is composed of three regions – the 5’ region anchors the primer to a particular location in the target, the 3’ region selectively amplifies the target sequence of interest and the Insert lies between the 5’ and 3’ regions, acting as a bridging structure which both (a) inserts target-independent sequence into the resulting amplicon and (b) increases the selective pressure of the 3’ region.

Unique features of PlexPrimers™ makes them suitable for:

1. Detection and discrimination of mutations or Single Nucleotide Polymorphisms (SNPs) in a multiplex reaction

  • The Insert sequence increases the stringency of amplification by the 3’ region, resulting in more specific amplification.
  • The PlexPrime™ amplicons are distinctly different by 12-15 bases, instead of 1 or 2 bases, which makes them easily identified in multiplex reactions.

2. Detection of a pool of nucleic acid sequences containing non-conserved regions

  • The Insert “skips” non-conserved regions, replacing the sequence and generating “generic” amplicons.
  • Equal and efficient amplification can then be achieved from these generic amplicons.

PlexPrime™ is a versatile tool that is compatible with challenging target sequences


Detection of SNPs and Mutations

During PCR, the complement of the Insert sequence (c-Insert) and the mismatch are incorporated into the PlexPrime™ amplicon.
PlexPrimePlexZymes1
Amplicons are detected in real time by allele-specific PlexZymes™. These are made up of two DNA oligonucleotides called “partzymes”, which assemble on the PlexPrime™ amplicons. Detection is specific for the PlexPrime™ amplicons as it includes the Insert sequence unique for the target allele.
PlexPrimePlexZymes3
PlexZymes™ catalyse cleavage of reporter probes, resulting in a fluorescent signal that corresponds to the presence of the allele.
PlexPrimePlexZymes3

Multiplexing mutation detection

The combination of PlexPrime™ and PlexZyme™ technology enables simultaneous detection of multiple variants, which is achieved by the use of allele-specific PlexPrimers™ and PlexZymes™. Each PlexPrimer™ is designed to have a 3’ region that matches the SNP or mutation, a distinct Insert sequence and a different 5’ region. This results in production of allele-specific amplicons which reduces primer competition and allows for allele-specific PlexZyme™ detection.

PlexPrimeMultiplexFig3

Figure 3: PlexPrime™ and PlexZyme™ strategy for multiplex detection of SNPs and mutations.


Detection of multiple serotypes or diverse species

Target organisms or group of organisms may have very diverse sequences and lack suitable regions for conventional probe design. In these cases, the PlexPrime™ can be used replace these variable regions with a unique sequence, improving the efficiency of amplification and detection.

The PlexPrime™ strategy has been applied for the detection of Enterovirus (Figure 6). Enterovirus are a large genus of RNA viruses consisting of numerous serotypes which can have highly variable sequences.

A single PlexPrimer™ is designed such that the 5’ and 3’ regions bind to the conserved sequence and the Insert skips 60 bases of highly variable sequence. The PlexPrime™ amplicon therefore replaces non-conserved region with the Insert sequence, which allows a single PlexZyme™ to detect all amplicons that are generated from the PlexPrimer™.

The Insert sequence of the PlexPrimer™ can be designed to “skip” the non-conserved region of the targets.
PlexPrimeDetection1
During PCR amplification, the Insert sequence replaces this non-conserved region, generating a generic amplicon.
PlexPrimeDetection2
The generic amplicon can then be detected with a single PlexZyme™ in real time.
PlexPrimeDetection3

 

Figure 6: Combination of PlexPrime™ and PlexZyme™ technologies detected various Enterovirus serotypes with similar efficiency despite large sequence differences in non-conserved regions.

PLexPrimeDetection4

References

Tan L. Y., Walker S. M., Lonergan T., Lima N. E., Todd A. V. Mokany E. 2017. Superior Multiplexing Capacity of PlexPrimers Enables Sensitive and Specific Detection of SNPs and Cluster Mutations in qPCR. PLOS One January 23rd.
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