Single-nucleotide polymorphism detection with "wire-like" DNA probes that display quasi "on-off" digital action

Abstract

Establishing a reliable genotyping protocol is a critical matter in post-sequence genetics. In this article, we describe a highly sensitive electrochemical detection of complementary DNAs (up to 43-mer) based on hole transport with molecular-scale, "wire-like" DNA probes. The presence of a single-base mismatch in the DNA duplexes caused a dramatic decrease in the electrochemical response. We applied this method to detect all of the possible transition and transversion SNPs and achieved "on-off"-type discrimination of fully complementary DNAs from their SNPs. Furthermore, naturally occurring polymorphisms, "hot spots" from the p53 gene, could clearly be distinguished from wild type by using our methodology.

Fig. 1.

A concept of our research. (Center) Chemical structure of HS-DNA-Fc. In the case of HS-DNA-Fc2, an ethane linker (-CH₂CH₂-) is substituted for the acetylene linker (-C≡C-). (Left and Right) Illustration for electrochemical discrimination of SNPs with a ferrocene-modified, wire-like DNA probe.

Fig. 2.

Electrochemical SNPs detection. Uncorrected SWV profiles at the gold working electrodes modified with HS-DNA-Fc·1 (fully matched duplex, red line), HS-DNA-Fc·12 (mismatched duplex, blue line), and HS-DNA-Fc2·1 (fully matched duplex, green line) (A) and the duplexes from HS-DNA2-Fc with fully matched DNA (red line), 248A SNP (light blue line), 249 SNP (black line), and 248T SNP (blue line) (B). Sequences are 5′-TGA ACC GGA GGC CCA T-3′ for fully matched DNA, 5′-TGA ACC AGA GGC CCA T-3′ for 248A SNP, 5′-TGA ACC GGA GTC CCA T-3′ for 249 SNP, and 5′-TGA ACT GGA GGC CCA T-3′ for 248T SNP (residues set in italics are the mismatched bases).

Fig. 3.

In situ detection on the electrode and electrochemical SNPs detection for longer target. (A) Relative current (I/I₀) plots at 0.31 V from SWV measurements for in situ detection (I₀ is the average current of HS-DNA-Fc·1 at 0.31 V). The first SWV measurements were carried out as described in Materials and Methods (point [a] for the target 1 and point [b] for the target 12). After rinsing three times with Milli-Q water (1 ml) at room temperature, the electrodes were subjected to the second SWV analyses under the same conditions (point [c]). Then, the electrodes were soaked in a fresh 50-μl target solution (200 μM 1 or 12), stirred at 25°C for 90 min with a thermomixer (24 × g), and rinsed with 1 M NaClO₄ (300 μl). The third measurements of the electrodes were carried out in 1 M NaClO₄ (points [d] and [e] for 1 and points [f] and [g] for 12). (B) SNP discrimination for long target strands (43-mer). Uncorrected SWV profiles at the gold working electrodes modified with HS-DNA-Fc·23 (fully matched duplex, red line) and HS-DNA-Fc·24 (mismatched duplex, blue line).