US Patent 10227632 Rolling circle amplification method

A method for performing a rolling circle amplification (RCA) reaction includes at least two rounds of RCA. The method includes providing a concatemeric first RCA product having a multiplicity of monomer repeats, each repeat representing a complementary copy of a first RCA template, wherein the nucleic acid molecule to be detected and/or analyzed, or its compliment, is contained in the first RCA template. The first RCA product is cleaved into monomer units which are reduced in size compared to the monomer repeat of the first RCA template. Monomer units resulting from the cleavage are circularized to form second RCA templates, which are smaller than the first RCA template. A second RCA reaction is performed on the second RCA template and a primer for the second RCA to form a second RCA product. The second RCA product, or a monomer unit derived from it, is detected or analyzed.