Mechanistic investigation of bead-based padlock rolling circle amplification under molecular crowding conditions.

We present a mechanistic investigation of bead-based padlock rolling circle amplification (RCA) under molecular crowding conditions, a sensitive and selective DNA detection method we have developed. Several important points to optimize the method were clarified: (i) the increase in the number of RCA products is proportional to the excluded volume of poly(ethylene glycol) (PEG), (ii) PEG facilitates ligation of padlock probe to form circular concatemers and monomers, both of which may act as a template for RCA, and (iii) hybridization of detection probe to the products may be facilitated at higher PEG concentrations.

Molecular crowding improves bead-based padlock rolling circle amplification.

Bead-based padlock rolling circle amplification (RCA), an ultrasensitive and accurate DNA detection technique, was conducted in a molecular crowding environment created by poly(ethylene glycol) (PEG). The number of RCA products generated increased and exhibited a bell-shaped dependence on PEG concentration. Experiments using magnetic beads suggested that facilitation of DNA ligation and hybridization is the main reason for the observed increase. Selectivity of the technique was retained in the presence of PEG. This technique is simple and can be utilized to detect target DNA with high accuracy and sensitivity in a variety of areas such as medical diagnosis and food analysis.

Bead-based Padlock Rolling Circle Amplification under Molecular Crowding Conditions: The Effects of Crowder Charge and Size.

Bead-based padlock rolling circle amplification under molecular crowding conditions, which we have developed for ultrasensitive detection of DNA, is examined to improve the detection efficiency and sensitivity of the method as well as to gain insight into the mechanism of the method. Both non-magnetic and magnetic sepharose microbeads were employed. Biotinylated DNA had to be pre-immobilized onto the microbeads in order to obtain products on the magnetic beads. The optimal concentration of biotinylated DNA was found to be about 5 µM, above which the number of products decreased. The effect of the crowder charge was examined, and neutral polymers were found to be effective on ligation and the hybridization step, while charged polymers were only effective on the hybridization step and inhibited the ligation and primer extension. The effect of the molecular weight of neutral dextran on the number of products was investigated, and the number of products was found to be increased with an increase in the molecular weight of dextran.

Development of a Simple Permeability Assay Method for Snake Venom-induced Vascular Damage.

We have developed a novel bioassay method for the detection of snake venom based on the permeability of endothelial cell monolayers cultured in Transwell cell culture inserts. This assay relies on the proteolytic degradation of capillary basement membrane proteins, a pathophysiological event that occurs due to snakebites in vivo. Transwell permeability assays with fluorescence measurements are advantageous with regard to ethical considerations for the use of animals. The assay time was reduced from 24 h for animal tests to 2 h, and many samples could be assayed easily.