Analytical Sciences

AFM-Imaging Diagnosis Method for Single Nucleotide Polymorphism Using Molecular Beacon DNA as an Intramolecular Ligation Template of Target DNA and a Viewable Indicator

Hisao YOSHINAGA,* Koji NAKANO,* Nobuaki SOH,** and Toshihiko IMATO*

*Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi, Fukuoka 819-0395, Japan
**Department of Agriculture, Saga University, 1 Honjo, Saga 840-8502, Japan

An AFM-imaging-based method for single nucleotide polymorphism (SNP) analysis is described. A stem-loop-forming 34-mer oligonucleotide (p34s) was designed. p34s contains the complementary sequence for K-ras (5′-GGT GGC-3′, t6G), one of the human oncogenes, at the 5′-end for target-recognition and five successive phosphorothioate linkages in the loop. The functional probe, either alone or hybridized with target DNA (p34s/t6G), relaxed upon treatment with “opener” DNA. The template/target DNA interstrand hybridization product is covalently connected by ligase if the correct target is used, but not hybridized species including mismatches. With these results, developed was a solid-phase SNP assay by transferring an aliquot of the product onto an Au(111) substrate for self-assembly, followed by AFM imaging. Clear contrasts that allow the detection of SNPs, were observed for the ligated and non-ligated species representing the loop-to-linear conformational change. Simple statistical surface-roughness analysis determined the lowest concentration of the sample to be 5 × 10⁻¹⁰ M, whose necessary sample quantity was 5 fmol.

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