Fig 1.
Detection of cancer DNA using novel enzyme-free assay.
The main steps of the LNA/DNA assay: 1) binding to gene-specific capture probe, 2) washing and cleavage from support, 3) adding signal-enhancing LNA/DNA probe and intercalating dye, 4) fluorescence detection. B = biotin, S = streptavidin, CPG = controlled pore glass, L = LNA.
Table 1.
Sequences and thermal denaturation temperatures of LNA/DNA capture probes prepared in this study.a
Fig 2.
Main principle of DNA detection by short LNA/DNA capture probes on solid support.
Target binding specificity results from the difference in melting temperature (Tm) between fully-matched and mismatched capture probe:target complexes. CPG = controlled pore glass.
Fig 3.
Detection of cancer DNA by fluorescence.
(A) Visualization of BRAF V600E mutation on solid-support containing capture probe CP2m: CP2m:HT29 (2.5 pM, tube 1), CP2m:LS411N (2.5 pM, tube 6). Signal is obtained under laboratory UV-vis lamp (excitation at 365 nm) at 19°C using 10 pM signal-enhancing probe and 0.6X EvaGreen dye. (B) Quantification of genomic BRAF targets by fluorometry in solution. Target titration curves were obtained for fully complementary and mismatched complexes (blue and red lines, respectively) of LNA/DNA capture probes CP2w and CP2m with corresponding targets and signal-enhancing probe P1: 5’-GCT A+GA CCA +AAA TCA CCT A+TT TTT ACT GTG AG+G TCT TCA TGA AGA +AAT AT-3’. LNA nucleotides are marked with plus before the corresponding letter.
Table 2.
Quantification of BRAF V600E mutation in cancer cell line DNA using fluorometry.a
Fig 4.
Fluorescence images of BRAF DNA fragments from cell line HT29.
(A) Complex of DNA with CP2m, signal-enhancing probe P1 and EvaGreen dye, bright dots with intensities over 80 are seen. (B) Target DNA re-annealed with CP2m and EvaGreen dye in the absence of P1, darker dots are seen with counts up to about 20. (C) EvaGreen dye in 1xX PBS (0.06X solution), no signal is seen. Images were obtained using two photon laser scanning microscope (ex 535+35nm; laser@ 840); at 19°C using 1.5 fM cancer DNA and re-annealing with 10 pM signal-enhancing probe and 0.06X EvaGreen dye. The images were taken using the same instrument settings and adjusted using the same intensity threshold. The graph below the images shows a line plot of the line in the image.