Synthesis and stretching of rolling circle amplification products in a flow-through system

Enzymatic isothermal rolling circle amplification (RCA) produces long concatemeric single-stranded DNA (ssDNA) molecules if a small circular ssDNA molecule is applied as the template. A method is presented here in which the RCA reaction is carried out in a flow-through system, starting from isolated surface-tethered DNA primers. This approach combines gentle fluidic handling of the single-stranded RCA products, such as staining or stretching via a receding meniscus, with the option of simultaneous (fluorescence) microscopic observation. It is shown that the stretched and surface-attached RCA products are accessible for hybridization of complementary oligonucleotides, which demonstrates their addressability by complementary base pairing. The long RCA products should be well suited to bridge the gap between biomolecular nanoscale building-blocks and structures at the micro- and macroscale, especially at the single-molecule level presented here.