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AssigneeConsejo Superior de Investigaciones Cientificas
InventorsLuis BLANCO DÁVILA, Miguel DE VEGA JOSÉ, Maria Isabel MARTINEZ JIMÉNEZ, Alicia DEL PRADO DIAZ
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The present invention relates to a method of primer-less isothermal amplification for detecting a target nucleic acids. In this method (a cartoon is shown in FIG. 16), a specific DNA padlock probe, complementary to the 3′ terminal region of a target nucleic acid, is circularized and amplified by rolling circle amplification (RCA), where the 3′-end of the target nucleic acid can act as an efficient primer by an optimized phi29 DNApol variant. The padlock-directed concatemeric ssDNA produced by RCA is further amplified by the combination of a modified phi29 DNApol (Qx3, Qx5) and a DNA primase (preferably TthPrimPol) which continuously synthesizes suitable DNA primers on the RCA product to trigger a hyperbranched rolling circle amplification (HRCA), that can be directly analyzed by any detection method. Variants of phi29 DNApol and their use in this and other amplification methods are also provided herein.
A method for detecting the presence of a single-stranded target nucleic acid by hyperbranched rolling circle amplification (HRCA), the method comprising the steps of:
The method according to any one of the previous claims, wherein the first and second regions of the target nucleic acid are located a maximum of 60 nucleotides of distance from the target nucleic acid's 3′ end.
The method according to any one of the previous claims, wherein the padlock comprises any one of SEQ ID NOs:
The method according to any one of the previous claims, wherein the single-stranded target nucleic acid is a ribonucleic acid.
A padlock probe comprising any of SEQ ID NOs:
A chimeric DNA polymerase comprising:
Dependencies are inferred from the claim text.