Linearity was determined using mixtures of plasmids carrying the wild-type or mutant sequence for the mutations GGT->GAT in codons 12 and 13 and the mutation CAA->CGA in codon 61 (see figure " Linearity of GGT->GAT mutation"). The plasmids were mixed in proportions to give four levels of mutation (5, 10, 30, and 50%). Each mixture was analyzed with three different lots of the therascreen NRAS Pyro Kit in three Pyrosequencing runs with three replicates each.
The results were linear within an allowable nonlinearity of 5 % units in the tested range of 5 to 50% mutation level. Similar results were obtained for the mutations GGT->GAT in codon 13 and CAA->CGA in codon 61.
The precision data allows the determination of the total variability of the assays and was obtained at three different levels by analysis of the above mentioned plasmid mixtures with three replicates each.
Repeatability (intra-assay and inter-batch variability) was calculated based on the data for determination of linearity (three runs on the same day using varying lots of the therascreen NRAS Pyro Kit). Intermediate precision (intra laboratory variability) was determined in three runs within one laboratory on three different days with varying operators, PyroMark Q24 instruments, and lots of the therascreen NRAS Pyro Kit. Reproducibility (inter-laboratory variability) was calculated from two runs each in an internal and external laboratory and using varying lots of the therascreen NRAS Pyro Kit.
The repeatability, intermediate precision, and reproducibility for the mutation GGT>GAT in codon 12 was 1.2–1.9, 1.0–2.0, and 1.3–3.1 % units, respectively, in the measured range of 5–50% mutation level. Similar results were obtained for the mutations GGT>GAT in codon 13 and CAA>CGA in codon 61.
Precision for the mutation GGT>GAT in codon 12
|% mutated plasmid|
Repeatability (Mean, SD)
Intermediate precision (Mean, SD)
Reproducibility (Mean, SD)
|5 ||7.5, 1.2||7.3, 1.0||6.7, 1.3|
|10||14.6, 1.3||13.5, 1.1||13.7, 1.3|
|30||37.8, 1.9||37.9, 1.5||36.1, 2.9|
|50||59.8, 1.7||60.4, 2.0||57.5, 3.1|