Clonal rolling circle amplification for on-chip DNA cluster generation

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.

[1]  G. Church,et al.  In situ localized amplification and contact replication of many individual DNA molecules. , 1999, Nucleic acids research.

[2]  J. McPherson,et al.  Coming of age: ten years of next-generation sequencing technologies , 2016, Nature Reviews Genetics.

[3]  Frank Diehl,et al.  Detection and quantification of mutations in the plasma of patients with colorectal tumors. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Ehrlich,et al.  Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency* , 1999, The Journal of Biological Chemistry.

[5]  G. Slater,et al.  Solid phase DNA amplification: a simple Monte Carlo Lattice model. , 2003, Biophysical journal.

[6]  H. Goodman,et al.  Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. , 1975, Journal of molecular biology.

[7]  G. Turcatti,et al.  Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms. , 2000, Nucleic acids research.

[8]  S. Odelberg,et al.  Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I. , 1995, Nucleic acids research.

[9]  Bin Li,et al.  Isothermal amplification method for next-generation sequencing , 2013, Proceedings of the National Academy of Sciences.

[10]  Lauren K. Wolf,et al.  Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison , 2006, Nucleic acids research.

[11]  Fredrik Dahl,et al.  Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments , 2005, Nucleic acids research.

[12]  S. Ehrlich,et al.  Replication slippage involves DNA polymerase pausing and dissociation , 2001, The EMBO journal.

[13]  Rameen Beroukhim,et al.  Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. , 2004, Nucleic acids research.

[14]  D. Dressman,et al.  Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[15]  David C. Thomas,et al.  Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction. , 2009, Archives of pathology & laboratory medicine.

[16]  P. Lizardi,et al.  Mutation detection and single-molecule counting using isothermal rolling-circle amplification , 1998, Nature Genetics.

[17]  Robert B. Hartlage,et al.  This PDF file includes: Materials and Methods , 2009 .

[18]  U Landegren,et al.  Padlock probes: circularizing oligonucleotides for localized DNA detection. , 1994, Science.

[19]  R. Murugan,et al.  Theory on the Mechanism of DNA Renaturation: Stochastic Nucleation and Zipping , 2015, PloS one.

[20]  Ronald W. Davis,et al.  Multiplexed genotyping with sequence-tagged molecular inversion probes , 2003, Nature Biotechnology.

[21]  Varshal K. Davé,et al.  Signal amplification by rolling circle amplification on DNA microarrays. , 2001, Nucleic acids research.