Multiplex Probe Amplification

Real-time PCR remains an important tool for detection and quantification of specific RNA or DNA sequences in samples. Real-time PCR is commonly multiplexed to allow the detection of a number of targets in one reaction. However, current probe-based methods allow detection of only one target sequence per fluorescence channel. In practice this limits simultaneous detection to five or six targets, depending on the real-time PCR platform.

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GeneFirst's Multiplex Probe Amplification (MPA) technology overcomes the limit on real-time PCR multiplexing and allows differentiation of up to six different targets per fluorescence channel.

The Principle
  • The MPA assay mixture contains PCR primers and probes for multiple targets. The mixture also contains a partially complementary oligonucleotide for each probe.

  • Real-time PCR is carried out in usual way to amplify the targets present. If a target is present the corresponding probe is consumed in the amplification stage.

  • Measuring the melting profile of the unconsumed probe, independent of hybridising to the target, is carried out after the amplification stage..

  • Each probe/partially complementary oligonucleotide complex has different characteristic melting temperature.

  • The absence of the characteristic melting profile peak of probes themselves (not with target) determines which probe has been consumed in the amplification stage and therefore what target is present in the starting sample.

Key Advantages
  • Multiplexed detection and differentiation of 15+ targets possible in a single tube

  • No specialised equipment required – compatible with commonly used real-time PCR platforms

  • Higher throughput than conventional real-time PCR possible thus enabling time savings and reduced reagent costs

  • Reducing the number of separate tests conducted on samples reduces risk of contamination and allows conservation of precious sample material.

Custom Assays available
We can design your assay according to your requirements using MPA technology.  Speak to us about your design needs today.
 
Licensing
If you'd like to know more about licensing opportunities with MPA, please contact us at licensing@genefirst.com

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