Synthesis of Circular Double-Stranded DNA Having Single-Stranded Recognition Sequence as molecular-Physical Probe for Nucleic Acid Hybridization Detection Based on Atomic Force microscopy Imaging

Abstract

A new class of DNA probes having a mechanically detectable tag is reported. The DNA probe, which consists of a single-stranded recognition sequence and a double-stranded circular DNA entity, was prepared by polymerase reaction. M13mp18 single strand and a 32mer oligodeoxynucleotide whose 5′-end is decorated with the recognition sequence were used in combination as template and primer, respectively. We have successfully demonstrated that the DNA probe is useful for bioanalytical purposes: by deliberately attaching target DNA molecules onto Au(111) substrates and by mechanically reading out the tag-entity using a high-resolution microscopy including atomic force microscopy, visualization/detection of the individual target/probe DNA conjugate was possible simply yet straightforwardly. The present DNA probe can be characterized as a 100%-nucleic acid product material. It is simply available by one-pod synthesis. A surface topology parameter, image roughness, has witnessed its importance as a quantitative analysis index with particular usability in the present visualization/detection method.

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

“…We found that, using this particular feature, simple histogram analysis could allow convenient quantification. 21,22 Figure 4 explains each frequency distribution fundamentally traces a specific curve typical of a normal distribution. Nonlinear curve fitting assuming a set of multiple Gaussian function peaks was also performed; the results are summarized in Table 2.…”