Probing of specific nucleic acids

Abstract

The present invention relates to improved methods for probing of specific nucleic acids using circularizable probes designed such that they report the presence of a target sequence by allowing a detectable moiety to remain bound if and only if the probe has been cyclized in a target-dependent linking reaction. The invention may be used for distinction between sequence specific varations of nucleic acids.

1 . A method of detecting a target nucleic acid sequence in a sample by contacting the sample with a detectable probe to hybridize the probe to the target sequence, and detecting the hybridized probe, said probe having two free nucleic acid end parts which are at least partially complementary to and capable of hybridizing to two at least substantially neighbouring regions of the target sequence, comprising the following steps: 
 a) hybridizing the probe ends to the target sequence under hybridizing conditions;    b) covalently connecting the ends of the hybridized probe with each other to form a circularized structure;    c) washing under denaturating conditions;    characterized in that the probe is provided with a cleavable or dissociable detectable function, and the method comprises the further steps of:    d) cleaving or dissociating said detectable function;    e) separating probes with connected ends from probes with non-connected ends by washing under denaturing conditions; and    f) detecting the presence and, if desired, location of the remaining probe as indicative of the presence of the target nucleic acid sequence.    
     
     
         2 . The method according to    claim 1   , characterized in that said detectable function is cleavable by cleaving a cleavable linker located on the same probe end as the detectable function.  
     
     
         3 . The method according to    claim 1    or    2   , characterized in that one or both of the probe ends have at least two branches, preferably with differential sequence specificities, and that a detectable function is provided on each of the branches on one end part of the probe, the detectable functions preferably being different and distinguishable from each other.  
     
     
         4 . The method according to    claim 3   , characterized in that one probe end is linear and the other probe end is branched, preferably bifurcated.  
     
     
         5 . The method according to    claim 1   ,    3    or  4 , characterized in that said detectable function is dissociable by being provided on a further circularizable probe hybridizing to said target-specific probe.  
     
     
         6 . The method according to    claim 1   ,    3    or  4 , characterized in that said detectable function is dissociable by being provided on said target-specific probe hybridizing to a further circularizable probe.  
     
     
         7 . The method according to any one of the preceding claims, characterized in that said target-specific probe is designed to hybridize to the target molecule to leave an interspace between the probe ends, that at least one additional probe is provided which is designed to hybridize to the target molecule in said interspace, and that the hybridized probes are covalently interconnected.  
     
     
         8 . The method according to any one of the preceding claims, characterized in that said target-specific probe or probes are designed to hybridize to the target molecule to leave a small gap between adjacent probe ends, and that said gap or gaps are filled by an extension reaction prior to covalently interconnecting the probe ends.  
     
     
         9 . The method according to any one of the preceding claims, characterized in that said covalent connection of the probe ends is performed by enzymatic, ribozyme-mediated or chemical ligation, preferably enzymatic ligation.  
     
     
         10 . The method according to any one of the preceding claims, characterized in that said target molecule is a DNA or RNA sequence.  
     
     
         11 . The method according to any one of the preceding claims, characterized in that probe or probes are oligonucleotides.  
     
     
         12 . The method according to any one of the preceding claims, characterized in that said probe or probes are immobilized to a solid phase.  
     
     
         13 . The method according to any of the claims  1 - 10 , characterized in that said target sequence is immobilized to a solid phase.  
     
     
         14 . A kit for detecting a target nucleic acid sequence in a sample, comprising 
 a) a probe having two free nucleic acid end parts which are at least partially complementary to and capable of hybridizing to two at least substantially neighbouring regions of the target sequence;    b) a means for connecting the ends to each other after hybridization to the target sequence;    characterized in    c) a cleavable of dissociable detectable function; and, optionally,    d) a cleaving agent.    
     
     
         15 . The kit according to    claim 14   , characterized in that the detectable function is a fluorophore, radiosiotope, hapten or enzyme.  
     
     
         16 . The kit according to    claim 14    or    15   , characterized in that one or both of the two free nucleic acid end parts of the probe have at least two branches, preferably with differential sequence specificities, and that each of the branches on one end part of the probe are provided with different detectable functions.  
     
     
         17 . The kit according to    claim 16   , characterized in that one probe end is linear and the other probe end is branched, preferably bifurcated.  
     
     
         18 . Use of the kit according to any one of    claims 14    to    17    for distinction between sequence specific variants of nucleic acids.  
     
     
         19 . A circularizable probe having two free nucleic acid end parts which are at least partially complementary to and capable of hybridizing to two at least substantially neighbouring regions of a target sequence, characterized in that the probe has a cleavable or dissociable detectable function on one of said end parts.  
     
     
         20 . The probe according to    claim 19   , characterized in that one or both of the two free nucleic acid end parts of the probe have at least two branches, preferably with differential sequence specificities, and that each of the branches on one end part of the probe are provided with different detectable functions.  
     
     
         21 . A circularizable probe having two free nucleic acid end parts which are at least partially complementary to and capable of hybridizing to two at least substantially neighbouring regions of a target sequence, characterized in that one or both of the two free nucleic acid end parts of the probe are branched, especially bifurcated.