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Overcoming the Limits of Conventional PCR

Molecular test technology amplifies target DNA/RNA by over a billion times providing sensitive and reproducible results.

Seegene has made important contributions to the field of multiplex PCR testing with the development of DPO™, TOCE™, and MuDT™ technologies. Employed across our various products, these innovations make it possible to detect from among multiple targets in a single PCR channel, increasing the total number of candidate targets in a single PCR reaction, compared to standard practice.

Dual Priming Oligonucleotide (DPO™)

DPO™ are primers made of two portions that work together to ensure highly specific hybridization, minimizing dimer formation. The DPO™ primers system is a highly accurate, rapid, and cost-effective method of primer binding. It allows for the detection of many targets in a single PCR reaction and is well-suited for high multiplex real-time PCR technology as it can overcome the limitations of conventional multiplex assays that include low plexing (4-6 targets) per reaction and reductions in specificity due to primer-dimer formation.

Benefits
  • Enables highly multiplex chemistries by minimizing non-specific amplification
  • Maximizes specificity and sensitivity
  • Prevents primer competition and non-target amplification where primer might bind to itself (hairpins) or to other primers (dimers). 
  • Withstands a wide range of annealing temperatures
  • Accurate, rapid, and cost-effective method of primer binding
  • Allows the detection of many targets in a single reaction
DPO™ overcomes the technical hurdles of conventional Multiplex PCR

CONVENTIONAL PCR

DPO™ PCR

Non-specific priming

Dual-specific priming

Decreased Specificity

No dimer formation – High Specificity

Decreased Sensitivity

High sensitivity

Difficulty in optimization

Tolerant to annealing temperature variation

Limitations in multiplexing

Reliable Multiple Target Amplification

Tagging Oligonucleotide Cleavage and Extension (TOCE™)

TOCE™ technology allows for the detection and differentiation of many targets in a single fluorescence channel using catcher-melting temperature analysis (CMTA). The key components for TOCE technology are a DPO primer (providing high specificity), a pitcher (tagging oligonucleotide which hybridizes specifically to the target region) and a catcher (dual-labeled artificial template). Up to 10 targets can be detected with five fluorescence channels in a single reaction (5 channels) with the same sensitivity as single-plex real-time PCR. TOCE™ provides accurate and reliable results under various assay conditions, when compared to the capability of conventional real-time PCR that requires more reactions to achieve high multiplex testing.

Benefits
  • High multiplex in a single channel
  • Up to 5 channels per reaction 
  • Multiple quality check steps for accuracy
  • Specificity is highly maintained across a wide range of annealing temperatures and in the presence of a high concentration of non-target DNA
Multiple Detection Temperatures (MuDT™)

MuDT™, multiple detection temperatures, enables determination of individual Ct values for multiple targets in a single detection channel. With MuDT™, a standard five channel system can detect up to 15 targets and their associated Ct values supporting both high multiplexing and quantification without additional melting curve analysis. MuDT™ technology combined with DPO™ and TOCE™ differ from conventional PCR where only a single Ct value is determined in each channel.

Benefits
  • Multiple Ct (threshold cycle) values in each fluorescence channel
  • Simultaneous detection, discrimination, and quantification of multiple targets in each channel of a single reaction
  • Each Ct value of multiple targets present in a sample is equivalent to that obtained by single target amplification
  • SNP genotyping in a single channel
Seegene’s Value for High Multiplex Testing

Comparison of PCR testing

Traditional PCR

Real time PCR

Seegene Multiplex PCR

Non-specific Amplification

Low to High

Low to High

Low – DPO

Plex / PCR channel (color)

1

1

2 – 3 / color

Plex / PCR reactions (targets)

4

4-6

12 to 18 – TOCE

# of CTs reported (targets)

       –

4-6

12 to 18 – MuDT

Potential for SNP detection

4

Low

High Up to 30 TOCE™ and MuDT™

Cost / reaction

++

+++

+

PCR plates per sample

Many

1 – 7

1

PCR runs / sample

Many

1 – 7

1

Samples / run

22 – 94

22 – 94

22 – 94

Seegene’s Value for Highly Multiples testing

Novaplex™ Assays are For Research Use Only. Not for Use in Diagnostic Procedures.

  • Novaplex™ Assays are For Research Use Only. Not for Use in Diagnostic Procedures.
  • Allplex™ 2019-nCoV Assay has an Emergency Use Authorization in the U.S.
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