Table 1.
Synthesized oligonucleotide sequences used in the experiments.
Figure 1.
Illustration of the label-free ssDNA detection strategy for detecting sequence-specific ssDNA target with its complementary ssDNA, S1 nuclease and DNA fluorescent dyes.
Figure 2.
Detection results of Sequence-specificity ssDNA in different surroundings.
(A). Fluorescence intensities of three viruses ssDNA and their hybridized products after S1 nuclease-mediated cleavage using SYBR Gold (1×). The concentration of target ssDNAs are 50 nM, and their complementary ssDNAs are 100 nM. a, H1N1-dsDNA; b, CaM-dsDNA; c, HCV-dsDNA; d, H1N1-ssDNA; e, CaM-ssDNA; f, HCV-ssDNA; g, blank. (B). Fluorescence intensities of three viruses ssDNA and their hybridized products in 4% pork muscle tissue homogenate after S1 nuclease-mediated cleavage using SYBR Gold (1×). The concentration of target ssDNAs are 50 nM, and their complementary ssDNAs are 100 nM. (C). Fluorescence intensities of 50 pM HCV-ssDNA and its hybridized products in 0.5 µg·mL−1 Lambda DNA standard solution after S1 nuclease-mediated cleavage using SYBR Gold (1×). (D). Fluorescence intensities of 50 nM HCV-ssDNA and its hybridized products in 5 µg·mL−1 Lambda DNA standard solution after S1 nuclease-mediated cleavage using SYBR Gold (1×).
Figure 3.
F/F0 values of three viruses and three types of dyes obtained from the proposed strategy.
The concentrations of target ssDNA and their complementary ssDNAs are 50 nM and 100 nM respectively, and the concentration of dyes is 1×. F0 represents the fluorescent intensity of solution blank containing S1 nuclease, HB solution and DNA dyes, and F represents the fluorescent intensity of the solution as described above upon addition of target ssDNA, or target ssDNA and its complementary sequence.
Figure 4.
Fluorescence intensity of oligonucleotides hybridized products with 1× PicoGreen after S1 nuclease-mediated cleavage.
The concentration of oligonucleotides and their complementary oligonucleotides is 50 nM and 100 nM respectively. (A) The effects of lengths of target ssDNA on fluorescence intensity. (B) The effects of nucleotide composition of target ssDNA on fluorescence intensity. (C) Linearity of the proposed method (three virus ssDNA). Plots of fluorescence intensity versus different concentrations of target ssDNA hybridized products (linear range, 50 pM–64 nM).
Figure 5.
Method selectivity for mismatches (H1N1, 1×SYBR Gold).
H1N1-1-G, H1N1-1-A: one base mismatch and the mismatched base is “G”, “A”, respectively. H1N1-2-CG, H1N1-2-AT: two bases mismatches and the mismatched bases are “CG”, “AT”, respectively. H1N1-3-CCG, H1N1-3-ATT: three bases mismatches and the mismatched bases are “CCG”, “ATT”, respectively. The concentration of all target ssDNAs is 50 nM, and their complementary ssDNAs is 100 nM.