Clonal rolling circle amplification for on-chip DNA cluster generation

• Published in: Biology methods and protocols Vol. 2; no. 1; p. bpx007
• Main Authors: Korfhage, Christian, Fricke, Evelyn, Meier, Andreas, Geipel, Andreas, Baltes, Mark, Krüger, Nadine, Herschel, Florian, Erbacher, Christoph
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.01.2017
• Subjects: Methods Manuscript; solid surface; forming DNA clusters; isothermal amplification
• ISSN: 2396-8923; 2396-8923
• DOI: 10.1093/biomethods/bpx007

Abstract Generation of monoclonal DNA clusters on a surface is a useful method for digital nucleic acid detection applications (e.g. microarray or next-generation sequencing). To obtain sufficient copies per cluster for digital detection, the single molecule bound to the surface must be amplified. Here we describe ClonalRCA, a rolling-circle amplification (RCA) method for the generation of monoclonal DNA clusters based on forward and reverse primers immobilized on the surface. No primer in the reaction buffer is needed. Clusters formed by ClonalRCA comprise forward and reverse strands in multiple copies tethered to the surface within a cluster of micrometer size. Single stranded circular molecules are used as a target to create a cluster with about 10 000 forward and reverse strands. The DNA strands are available for oligonucleotide hybridization, primer extension and sequencing.