A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (∼60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays.

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By means of scanning tunneling microscopy (STM), we succeeded for the first time in the quantitative analysis of the intramolecular conformation of a supramolecule, cyclodextrin (CyD) necklace, driven by hydrogen bonding. Contrary to the current model, based on macroscopic analyses, which indicates that all CyDs are arranged in head-to-head or tail-to-tail (secondary-secondary or primary-primary hydrogen bonding) conformation, about 20% head-to-tail (primary-secondary hydrogen bonding) conformation was found to exist in the molecule. In addition, comparing the STM results with the theoretical model of the necklace formation, the formation ratio of the tail-to-tail and head-to-tail conformations due to the strength difference between primary-primary and primary-secondary hydrogen bonds of CyDs was directly obtained, for the first time, to be 2:1.