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

作者：Yoshinaga Hisao; Nakano Koji^*; Soh Nobuaki; Imato Toshihiko

来源：Analytical Sciences, 2012, 28(10): 939-945.

DOI：10.2116/analsci.28.939

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%26apos;-GGT GGC-3%26apos;, t6G), one of the human oncogenes, at the 5%26apos;-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 %26quot;opener%26quot; 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 x 10(-1) M, whose necessary sample quantity was 5 fmol.

• 出版日期2012-10