The iPLEX assay for SNP Genotyping is the leading application for fine mapping SNP genotyping. Recently, a new protocol has been developed on the basis of the iPLEX assay to improve the multiplexing capabilities of the assay up to a level of 40-Plexing. This new protocol is named iPLEX Gold.
The iPLEX assay is based on a simple single-base primer extension assay. A protocol overview is provided in Figure 1.
The iPLEX assay is truly homogeneous, in the sense that after PCR, reagents are added to the reaction cocktail in a three step protocol. No transfer or pipeting into different reaction containers is required.
PCR primers are designed in a region of approximately 100 base pairs around the SNP of interest and an extension primer is designed immediately adjacent to the SNP. The assay design is performed in a highly automated fashion by the AssayDesigner Software module.
The starting point of the iPLEX assay is PCR amplification, followed by the addition of Shrimp Alkaline Phosphatase (SAP) to inactivate remaining nucleotides in the reaction. Following a brief incubation, the primer extension mixture is added and conducted using a standardized cycling program. Finally, CleanResin is added to the mixture to prepare it for deposition on a 384 well SpectroChip®. SpectroChips enable an automated readout and data analysis by a Compact™MALDI-ToF mass spectrometer.
Data analysis is performed on the Typer Software Module, a screenshot of the graphical user interface is provided in Figure 2.
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