STAR* (Scalable Target Amplification Routine) technology is a method for simultaneous quantitative measurement of multiple target nucleic acids. STAR technology is based on novel functional integration of Polymerase Chain Reaction (PCR) and capillary electrophoresis (CE). In typical STAR assay amplification of multiple nucleic acid targets is monitored by sampling PCR reaction at sequential PCR cycles, separating and quantifying PCR products in sequential samples by CE to reconstruct amplification curves and use them for simultaneous quantification of multiple nucleic acid targets.
Similar to real-time PCR amplification, a threshold cycle Ct (cycle at which measured amount of PCR product crosses pre-selected value) is determined from amplification curve for each of the targets in the exponential phase of PCR amplification. Standards of known quantity for each of the targets can be used in separate experiments to establish calibration curve of measured Ct values as a function of log of standards concentration. The measured Ct value for unknown sample can be determined using the created calibration plot. An alternative quantification algorithm, unique for STAR technology, can use multiple quantitative standards included in each assay for creation of internal calibration plot for each individual STAR assay.
STAR assays can be performed as a discontinuous process, in which small aliquots of PCR reaction mixture collected at subsequent cycles are temporary stored in the receiving plate/s, followed by analysis using standalone capillary electrophoresis instrument. This approach, STACE (Semi-automated Thermal cycler And CE) is currently implemented at Primera. Detailed description of STAR process on STACE system is presented in the corresponding section.
Alternatively, sampling of PCR reaction and CE analysis can be performed as an integrated process, in which PCR amplification takes place concurrently with CE analysis. The integrated approach requires a novel instrument system, ICE (Integrated CE) currently under development at Primera. This instrument combines two functional modules: PCR thermal cycler (amplification module) and CE (analysis module) to perform direct electrokinetic injection from PCR reactions into the separating capillaries. The simplified instrument architecture will result in the smaller, bench-top unit fitting in the research and clinical environment.
* For Research Use Only. Not for use in diagnostic procedures.