We describe conditions for rolling-circle amplification (RCA) of individual DNA molecules 5–7 kb in size by >109-fold, using 29 DNA polymerase. The principal difficulty with amplification of small amounts of template by RCA using 29 DNA polymerase is ‘‘background’’ DNA synthesis that usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while k...

Recently, we developed a simple isothermal nucleic acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), to detect specific DNA sequences with great sensitivity and large dynamic range. In this paper, we combined PG-RCA with a three-way junction (3WJ) formation, and detected specific RNA molecules with high sensitivity and specificity in a one-step and isothermal...

Hypochlorous acid (HClO) is a common reactive oxygen species (ROS), with high chemical reactivity. Myeloperoxidase (MPO) an enzyme that catalyzes in vivo redox reactions between H2O2 and Cl− to produce HClO. Abnormal levels of HClO MPO may lead oxidative stress, irreversible tissue damage and, thus, serious diseases; they are thus becoming important biomarkers therapeutic targets. In this work,...

Kühnemund, M. 2016. Single Molecule Detection. Microfluidic Automation and Digital Quantification. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1189. 57 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9498-8. Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, ...

Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydrophobic alkane COP was modified to have carbonyl functionalities through oxygen plasma and chemical etching treatments to increase usefulness for chemical and biochemical applicatio...

A nucleic acid amplification technique termed as netlike rolling circle amplification is proposed by introducing a nicking enzyme into the existing hyperbranched rolling circle amplification system. Surprisingly dense and uniform network morphology is observed; and cubic amplification is achieved for the sensitive detection of a sequence from HIV.

We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using 29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and 29 DNA...

In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL) detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease)-assisted target recycling amplification (TRA), rolling circle amplification (RCA) and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probe...