Fig 1.
Detecting NPM1 gene mutations in patient samples using a standard PCR assay.
The PCR products are visualized on 10% polyacrylamide gel. MT indicates NPM1 mutated control and WT indicates NPM1 wild-type control. A double band indicates a heterogeneous mutation.
Fig 2.
HRM difference curves of Tc determinations.
(a) The height of the difference curve for mutants increased with increasing Tc from 80–90°C. (b) No signals of wild-type products were produced when increasing the Tc from 80–88°C, while the wild-type was amplified when increasing the Tc to more than 88°C and presented the signal in the HRM difference curve.
Fig 3.
The E-ice-COLD-PCR assay combined with HRM analysis of four types of NPM1 mutations.
Following the E-ice-COLD-PCR assay, the PCR products were analyzed by HRM analysis. The HRM difference curves show the peaks of NPM1 mutation types A, B, D, and J, while no peak in the NPM1 wild-type indicated that the E-ice-COLD-PCR assay can inhibit wild-type amplification. Meanwhile, it can enhance the amplification of the four types NPM1 mutations.
Fig 4.
LOD of the E-ice-COLD-PCR assay combined with HRM analysis.
Following the E-ice-COLD-PCR assay, the PCR products were analyzed by HRM analysis. The HRM difference curves show the height of the curves decreased with the decreasing percentage of mutants, and the peak disappeared with the decreasing percentage of mutants from 6.25% to 0%.
Fig 5.
Detecting NPM1 gene mutations by HRM analysis.
Detecting NPM1 gene mutations using the E-ice-COLD-PCR assay and generating the data into HRM difference curves. Red line indicates mutant control. Green line indicates wild-type control. The wild-type samples present the peak at the same location as the wild-type control, and the mutant samples present the peak at the same location as the mutant control.
Fig 6.
Direct sequencing in the reverse direction of mutated products.
(a) Mutated products from the standard PCR assay (b) Mutated products from the E-ice-COLD-PCR assay. The results showed that the NPM1 mutation type A is a 4 bp insertion.