Developing the Assay
DxS produces real-time PCR assays that yield results in less than three hours from DNA extraction, and can detect down to 1% mutation in a background of wild-type DNA. The kits contain a standard (containing synthetic templates of all the mutations detected in the kit, which is used to show that each assay is working efficiently), a control assay (to assess quantity and quality of the DNA sample, this is an ARMS and Scorpions reaction that amplifies a region in the gene of interest that is not affected by mutations), and mutation assays (individual ARMS and Scorpions primers designed to only detect the mutation screened for by that kit).
Each assay (control and mutation) is multiplexed with an exogenous control to ensure the reaction is working efficiently and to allow detection of any inhibitors. In a real-time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The DxS kits use ARMS for discrimination and Scorpions for detection.
ARMS technology is based on the principle that primer extension is efficient when the 3´ terminal base of a primer matches its target, whereas extension is inefficient or nonexistent when the terminal base is mismatched. Primers are designed against the mutation of interest, and an amplicon is produced only if the mutation is present.
Scorpions probes are bifunctional molecules that contain a PCR amplification primer attached to a probe sequence. During PCR (Figure 1), the primer is extended to form an amplicon, and the probe, which contains an amplicon-specific sequence, binds to its target to produce a fluorescent signal. The amount of fluorescence will be proportional to the amount of amplicon generated.
The combination of ARMS and Scorpions technologies in a real-time PCR assay are ideal for mutation detection in tumor samples. This is because tumors are heterogenic, in that when the biopsy is taken there will be tumor cells and normal healthy cells, but the mutation may only be present in a few cells of the tumor biopsy.
This means that there will be limited material available that will contain the mutation. The combination of ARMS and Scorpions allows 1% mutant to be detected in a background of wild-type, meaning it is more likely to detect the mutation compared to sequencing, which has a sensitivity of approximately 20%.
ARMS and Scorpions technologies are used to obtain Ct values in real-time PCR. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceed background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level the greater the amount of target nucleic acid in the sample).
The assays work on delta-Ct values, which is the difference between two Ct values. A control assay is run with every sample that produces a Ct value for the amount of the gene present in the sample. A separate Ct value is produced from the specific mutation assay, and the delta-Ct is the difference between the mutation Ct and the control Ct. Cut-off limits have been validated, and the delta-Ct from the sample is compared to the cut-off value, if the value is equal to or less than the cut-off then the sample is deemed positive for that mutation, if the delta-Ct is greater than the cut-off value the sample is negative or beyond the limits of the kit. Figure 2 shows typical results with the PI3K assay.
The ARMS and Scorpions primers were designed for the specific regions in the PIK3CA gene that will be detected by the assay. The format of DxS’ other kits were followed.