Nanoparticle-based detection and quantification of DNA with single nucleotide polymorphism (SNP) discrimination selectivity

Abstract

Sequence-specific DNA detection is important in various biomedical applications such as gene expression profiling, disease diagnosis and treatment, drug discovery and forensic analysis. Here we report a gold nanoparticle-based method that allows DNA detection and quantification and is capable of single nucleotide polymorphism (SNP) discrimination. The precise quantification of single-stranded DNA is due to the formation of defined nanoparticle-DNA conjugate groupings in the presence of target/linker DNA. Conjugate groupings were characterized and quantified by gel electrophoresis. A linear correlation between the amount of target DNA and conjugate groupings was found. For SNP detection, single base mismatch discrimination was achieved for both the end- and center-base mismatch. The method described here may be useful for the development of a simple and quantitative DNA detection assay.

Figure 1.

DNA sequences used in this study. For Strands A′ and revA′, the underlined sequences are the recognition site of EcoR V and the arrows indicate the enzyme cleavage site. For the mismatched DNA sequences, the underlined base is the mismatch. SM1/SM2/SM3/SM4/SM53/SM6 refer to single-base mismatched DNA, DM refers to double-base mismatched DNA and NC refers to non-complementary DNA.

Figure 2.

Schematic picture of the formation of nAu–DNA conjugate groupings.

Figure 3.

Formation of nAu–DNA conjugate dimers with various lengths of DNA target. Lane A corresponds to nAu without Strand A or revA modification (control); Lane B corresponds to nAu–A + nAu–revA conjugates with no target; Lanes C, D, E and F correspond to nAu–A + nAu–revA conjugates with 26, 24, 22 and 20 bases of target DNA respectively.

Figure 4.

Grouping percentage of nAu–DNA conjugates with various ratios of target DNA. Lanes G–O correspond to nAu–A: nAu–revA: target DNA ratio of 1:1:1, 1:1:0.8, 1:1:0.5, control (nAu without Strand A/revA conjugation), 1:1:0, 1:1:0.3, 1:1:0.25, 1:1:0.15 and 1:1:0.1, respectively. Gel picture shows the combined results from two experiments.

Figure 5.

Grouping percentage of nAu–DNA conjugates with different ratios of target DNA.

Figure 6.

Grouping percentage of nAu–DNA conjugates with different ratios of target DNA. Lanes P–T correspond to nAu–A and nAu–revA with target DNA at a ratio of 2:1:2 (nAu–A:nAu–2x revA:target DNA), 2:1:5, control (nAu without Strand A/revA conjugation), 2:1:20 and 2:1:100, respectively. Gel picture shows the combined results from two experiments.

Figure 7.

TEM image of nAu–DNA conjugate dimers.

Figure 8.

SNP discrimination using nAu–DNA conjugates and 24-base target DNA. Lanes U-AA correspond to, nAu–A and nAu–revA plus 24-base perfectly matched DNA (PM), single base matched DNA (SM1/SM2/SM3), double base matched DNA (DM), non-complementary DNA (NC) and no target DNA, respectively.

Figure 9.

SNP discrimination using nAu–DNA conjugates and 26-base target DNA. Lane BB corresponds to nAu–A and nAu–revA and 26-base perfectly matched DNA (PM) plus single base matched DNA SM4, SM5 and SM6 (ratio of nAu–A:nAu–revA:PM/SM4/SM5/SM6 = 1:1:0.25/0.25/0.25/0.25), and Lanes CC to EE correspond to nAu–A and nAu–revA plus SM4/SM5/SM6, respectively (ratio of nAu–A:nAu–revA:SM DNA = 1:1:1).