Probing of specific nucleic acids

A method, wherein a target nucleic acid sequence is detected, comprising the following steps:

• a) providing an oligonucleotide probe immobilized to a solid support, the immobilized probe comprising two end parts having at least one 3′-end sequence and at least one 5′-end sequence, wherein one of said end parts is provided with a solid phase anchor by which the probe is immobilized to the support and wherein the other end part comprises at least one detectable function provided on a dissociable part of said probe,
• b) contacting the immobilized probe with a target nucleic acid sequence allowing the 3′-end and the 5′end of the immobilized probe to hybridize to at least substantially neighboring regions of said target nucleic acid sequence under hybridizing conditions;
• c) covalently connecting the ends of the hybridized oligonucleotide probe to each other to form a circularized structure;
• d) dissociating said dissociable part;
• so that if said probe is circularized in step c), the probe part comprising the detectable function becomes connected by catenation to the other probe part comprising the solid phase anchor and thereby connected to the support, and cannot be de-connected from the immobilized probe by said dissociating;
• but wherein if said probe is not circularized in step c), the probe part comprising the detectable function is not connected by catenation to the other probe part comprising the solid phase anchor, and hence is not connected to the support, and can be de-connected from the immobilized probe by said dissociating;
• e) separating non-connected detectable functions from the solid support by washing under denaturing conditions;
• f) detecting the target nucleic acid sequence by detecting the presence, and optionally, quantity and/or location of the connected detectable function, and
• wherein said probe comprises two padlock probes and said dissociable part is one of said padlock probes, which hybridizes to the target nucleic acid sequence in step (b) and to the other padlock probe carrying said solid phase anchor.

A method, wherein a target nucleic acid sequence is detected, comprising the following steps:

• a) providing an oligonucleotide probe, said probe comprising two end parts having at least one 3′-end sequence and at least one 5′-end sequence, wherein one of said end parts is provided with a solid phase anchor for immobilization of said probe to a solid support and wherein the other end part comprises at least one detectable function and a cleavable site which lies between the detectable function and the solid phase anchor,
• b) contacting the probe with a target nucleic acid sequence allowing the 3′-end and the 5′end of said probe to hybridize to at least substantially neighboring regions of said target nucleic acid sequence under hybridizing conditions;
• c) covalently connecting the ends of any hybridized oligonucleotide probe to each other to form a circularized structure;
• d) immobilising said probe to a solid support by means of said solid phase anchor (i) before step b) or (ii) between steps c) and e);
• e) cleaving the circularized and non-circularized oligonucleotide probe at the cleavable site between the detectable function and the solid phase anchor;
• so that if said probe is circularized in step c), the probe part comprising the detectable function becomes covalently connected to the other probe part comprising the solid phase anchor and thereby connected to the support, and cannot be de-connected from the immobilized probe part by said cleaving;
• but wherein if said probe is not circularized in step c), the probe part comprising the detectable function is not covalently connected to the other probe part comprising the solid phase anchor, and hence is not covalently connected to the support, and can be de-connected from the immobilized probe part by said cleaving;
• f) separating non-connected detectable functions from the solid support by washing under denaturing conditions;
• g) detecting the target nucleic acid sequence by detecting the presence, and optionally, quantity and/or location of the connected detectable function, and
• wherein said cleavable site is a disulphide or a deoxyuridine residue or a peptide residue or a nucleotide sequence susceptible to cleavage by endonuclease, wherein in step e), said cleaving of the oligonucleotide probe takes place using a cleaving agent being a reducing agent, a uracil DNA glycosylase, a peptidase or an endonuclease, respectively.