Fig 1.
(A) In the case of KRAS wild-type allele, the specific wild-type reporter hybridizes in solution with the single strand PCR product (ssPCR), whereas the specific mutant reporter (with the variant position circled and in red) does not. (B) Different barcodes in the 3’ domain of the reporter sequences (Barcode W for wild-type, Barcode M for mutation) direct the ssPCRs to different position on the array. The position in the array is revealed when the U-tag sequence at 5’-end of the ssPCR interacts with the complementary Cy3-labeled oligonucleotide, Universal-Cy3 (U-Cy3), added in the last step of the assay.
Fig 2.
Examples of genotyping of KRAS, NRAS and BRAF mutations.
A) Schematic representation of the spotted barcode probe array. Silicon chips coated with copoly(DMA-NAS-MAPS) are used as substrates for the covalent attachment of amino-modified barcode probe oligonucleotides arrayed at discrete locations. Each position in the grid identifies an individual barcode probe address corresponding to KRAS codon 12–13, KRAS codon 61 (Q61H1 c.183A>C, Q61H2 c.183A>T), KRAS codon 146, NRAS codon 12–13 and BRAF mutations. The light grey portion of the array is spotted with an amino-modified oligonucleotide (COCU8), not correlated with the genes, to be used as reference spots. B) Microarray scanning of the Cy3 fluorescence signal of five different silicon chips. Each robotically spotted array is hybridized with an individual single strand PCR incubated with the whole set of dual-domain reporters. The fluorescence detection is obtained incubating the array with a mixture of the universal Cy3 labeled oligonucleotide complementary to the tagged-reverse primer of the single strand PCR and with a Cy3-labeled oligonucleotide (COCU10) complementary to COCU8. KRAS G12S, KRAS Q61R, KRAS A146T, NRAS G13R and BRAF V600E correspond to the control sample containing the indicated mutation. All the five samples of known genotype (mutant homozygous or heterozygous in case of KRAS G12S and BRAF V600E) were correctly identified.
Fig 3.
A) Picture showing the setup utilized to realize the calibration curves for the KRAS codon 12–13 mutations. B) Microarray scanning of the Cy3 signal of the coated silicon slide and the magnification of a portion of it showing the result of the hybridization of four different concentration of mutant DNA in four different wells. C) Plot representing the relative fluorescence intensity of the signal corresponding to the Cy3-labeled mutated single strand PCR bound to the G12S barcode probe. The points, calculated as the average of the intensity of four spots, correspond to the percentage of the KRAS G12S mutation in a background of KRAS wild-type DNA. The value at point 0 represents the relative fluorescence intensity of the background presents on the G12S barcode probe array of the well hybridized with wild-type control sample. The error bars are the standard deviations of the fluorescence intensity of each well. The equation of the trend line of the graph is utilized to extrapolate the limit of detection (LOD) for the assay.
Table 1.
The extrapolated limits of detection for the seven most common mutations of the KRAS gene.
Fig 4.
Analysis of DNA extracted from Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples.
The spotting schema of the barcode sequences is the same of Fig 2A. (A) Cy3 fluorescence image and the plot of the relative fluorescence intensity of the sample identified with KRAS G12S mutation. (B) Cy3 fluorescence image and the plot of the relative fluorescence intensity of the sample identified with KRAS Q61H1 (c.183A>C) mutation. (C) Cy3 fluorescence image and the plot of the relative fluorescence intensity of the sample identified with NRAS G12V mutation. The yellow squares in the images were used to highlight more easily the analyzed spots. The bars are the average of the intensity of the 4 spots (2 X 2 subarray) of each barcode probe subarrays. The error bars are the standard deviations of the fluorescence intensity of each sample. Q61H1 c.183A>C, Q61H2 c.183A>T.
Fig 5.
Analysis of ctDNA extracted from plasma of 4 patients with metastatic colorectal cancer.
A) Microarray scanning of the Cy3 fluorescence signal of four different plasma samples. Only the part of the array corresponding to the barcode probes for KRAS codon 12 and 13 is shown. In Plasma1 the barcode probes corresponding to KRAS G12D and G12R mutation are highlighted. B) Schematic representation of the spotted barcode probe array. Q61H1 c.183A>C, Q61H2 c.183A>T C) Microarray scanning of the Cy3 fluorescence signal of the BRAF codon 600 area corresponding to the BRAF barcode probes for the Plasma 2 sample. The frame highlights the barcode probes corresponding to the BRAF V600E mutation.