PROCESS OVERVIEW

Based on the information provided by our partners, Asper will start working on the design of the custom assay.

As the first step, the design of APEX and PCR primers is performed. 25mer APEX primers will be designed and verified by using specialized software ProbeDesignerTM (developed by Asper Biotech`s daughter company BioData Ltd.) that alerts for possible secondary structure effects. PCR primers will be designed using another software package BioPrimerTM that allows us to select the primers. In the second stage run the program GenomeTestTM will be run to insure that the selected PCR primers will produce a unique product. Chip TesterTM software helps to ensure that the PCR primers do not hybridaze to APEX primers and that each APEX primer covers only one unique site in the PCR product mix. The designed primers will be ordered from an external provider, who ensures the highest quality of the synthesis. 
  
Once the oligos have been synthesised, Asper will design the appropriate layout and develop the chip. Sometimes our clients would like to see specific SNPs to be located on the chip together, however routinely we are using a randomized approach. 
  
Based on the layout of the chip the APEX primers will be diluted and spotted onto a small set of test-slides, where the first test reactions and the following protocol optimization are executed
and spotted onto a small set of test-slides, where the first test reactions and the following protocol optimization are executed.
 
PCR conditions will be optimized and if possible multiplexing will be introduced. The fragmentation and purification protocols will be tested and the best combination will be used in the later processes. In order to gain the best results out of the APEX reaction, the primer extension reaction will be tested by using different fluorescent dye combinations and enzyme combinations. 
 
The protocol optimizations will be followed by the validation phase I, where the initial testing of the chip assay will be carried out. Approximately 10 different DNA samples will be used. 
 
Based on the results of the validation phase I we can evaluate, whether there is a need to redesign a few primers. In this stage we will analyze carefully all the signals and replace some primers for the positions, that might yield inconsistent results. 

In case there was a need to redesign some of the primers they will be diluted and spotted to a new array. The chips will be validated according to the protocols used in the phase I. However after the validation phase I has been completed, the phase II validation could be introduced. In case of mutation analysis the phase II will be used to evaluate each mutation detection on the chip with DNA samples carrying the specific mutation in it. In particular for the mutation detection chips Asper has introduced also a phase III validation, that includes different ways of blind testing or specific titrations, which give us a better insight into how well the new chip works.

In the end, a detailed report on the results and the process of the assay development will be given to the customer.