Primer generation rolling circle amplification

摘要

A method of amplifying a nucleic acid includes combining a first linear primer with a polymerase and a circular probe. The circular probe contains at least one antisense sequence to a second nucleic acid sequence and at least one antisense sequence to the first linear primer. The method also includes producing at least one repeat of an antisense copy of the circular probe by rolling circle amplification. The antisense copy contains at least the second nucleic acid sequence. The method further includes generating more than one second linear primers from each copy of the second nucleic acid sequence; and hybridizing the second linear primers to more than one of the circular probes. A ribbon probe includes a circular probe and a lock probe. The lock probe contains at least a cleavable linker, and the circular probe and the lock probe are unable to dissociate without cleaving the cleavable linker.

主权项

1. A method of amplifying a nucleic acid, comprising:
(a) providing a first linear nucleic acid primer; (b) combining said first linear nucleic acid primer with a polymerase and a circular nucleic acid probe, wherein said circular nucleic acid probe contains at least one antisense sequence to a second nucleic acid sequence and at least one antisense sequence to said first linear nucleic acid primer; (c) producing at least one repeat of an antisense copy of said circular nucleic acid probe by rolling circle amplification using said polymerase, wherein said antisense copy contains at least said second nucleic acid sequence; (d) generating a plurality of second linear nucleic acid primers from each copy of said second nucleic acid sequence in each antisense copy by hybridizing a nucleic acid molecule to said second nucleic acid sequence in each antisense copy, by
(i) cleaving said antisense copy and/or said nucleic acid molecule by a cleavage agent, thereby generating precursors of said second linear nucleic acid primers capable of self-priming;(ii) self-priming said precursors of said second linear nucleic acid primers; and(iii) extending the self-primed precursors by
(1) polymerase followed by cleavage with said cleavage agent, or(2) RNA polymerization, thereby generating said plurality of second linear nucleic acid primers; (e) hybridizing said second linear nucleic acid primers to a plurality of said circular nucleic acid probes; and (f) repeating steps (c), (d) and (e) at least once.