AFM-imaging diagnosis method for single nucleotide polymorphism using molecular beacon DNA as an intramolecular ligation template of target DNA and a viewable indicator.
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.
Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Fukuoka 819–0395, Japan.
Hisao
H
Yoshinaga
Koji
K
Nakano
Nobuaki
N
Soh
Toshihiko
T
Imato
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D007202:Indicators and Reagents / Q000737:chemistry; D055029:Inverted Repeat Sequences; D018625:Microscopy, Atomic Force / Q000379:methods*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D015342:DNA Probes; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D004247:DNA