PlexPrime™ is a novel method for nucleic acid amplification that creates amplicons which are distinctly different from the parent sequence. The PlexPrimer™ is composed of three regions (Figure 1). 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 and acts 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.
Figure 1: PlexPrimers™ are composed of three distinct regions: (i) 5′ region, (ii) an Insert sequence, and (iii) 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 PlexPrimer™ 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 can be used to “skip” non-conserved regions, for example when targeting multiple strains of bacteria or viruses.
When common Insert replaces the non-conserved region general amplicons are created.
- 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
Multiplexing mutation detection
The combination of PlexPrime™ and PlexZyme™ technology enables simultaneous detection of variants, which is achieved by the use of allele-specific PlexPrimer™ 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.
Figure 3: PlexPrimer™ and PlexZyme™ strategy for multiplex detection of SNPs and mutations.
Multiplex detection of point mutations with high sensitivity and specificity
The combination of the PlexPrime™ and PlexZyme™ technology were used to detect 13 mutations in codon 12, 13 and 61 of the RAS gene in a single multiplex reaction (Figure 4). Detection of these mutations was achieved on a conventional qPCR machine by stacking each Universal Probe read-out to more than one mutation. Figure 4 illustrates the 13-plex reactions in 2 wells. Well 1 was an 8-plex reaction containing 3 Universal Probes to be read in 3 fluorescence channels. Signal from the first (blue), second (yellow) and third probe (green) corresponded to detection of 1, 4 and 3 mutations, respectively. Well 2 contained a 5-plex reaction read by a single Universal Probe (purple). The technologies enabled detection of all mutants with sensitivity of ≤1% in the background of wild type DNA.
Figure 4: Mass multiplexing of RAS gene mutations using PlexPrime™ and PlexZyme™ technology. Detection was achieved on conventional qPCR machine with reaction sensitivity of ≤1%.
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™.
Figure 5: The use of a single PlexPrime™ and PlexZyme™ to enable detection of diverse groups of organisms containing highly variable sequences.
Efficient amplification of multiple Enterovirus serotypes
Figure 6: Combination of PlexPrime™ and PlexZyme™ technologies detected various Enterovirus serotypes with similar efficiency despite large sequence differences in non-conserved regions.