Nucleic acid detection method

Abstract

The present invention relates to methods for the detection of nucleic acids of defined sequence, and compositions and kits for use in said methods. The methods employ nicking agent(s) and a sequential series of oligonucleotide probes to produce probe fragments in the presence of a target nucleic acid.

1 .- 58 . (canceled) 
     
     
         59 . A kit for detecting the presence of a target nucleic acid of defined sequence comprising the following:
 (a) a first oligonucleotide probe [X] comprising:
 i. a complementarity region [A] capable of sequence specific hybridisation to the target nucleic acid to form double-stranded nucleic acid that is specifically recognised by a nicking agent and contains a cleavage site for said nicking agent; and 
 ii. a further complementarity region [B] which is separated from complementarity region [A] following cleavage at said cleavage site to produce a first probe fragment, and which is capable of sequence specific hybridisation to a second oligonucleotide probe [Y] to form a double-stranded nucleic acid that is specifically recognised by a nicking agent, and contains the anti-sense sequence of the cleavage site for said nicking agent; 
   (b) the second oligonucleotide probe [Y] comprising:
 i. a complementarity region [C] capable of sequence specific hybridisation to the complementarity region [B] in said first oligonucleotide probe to form a double-stranded nucleic acid that is specifically recognised by a nicking agent and contains a cleavage site for said nicking agent; and 
 ii. a second region [D] which is separated from complementarity region [C] following cleavage at said cleavage site to produce a second probe fragment, and which is not capable of sequence specific hybridisation to the complementarity region [A] or [C] of the first or second oligonucleotide probes to form a site that is specifically recognised and cleaved by any of the said nicking agents; 
   (c) optionally a third oligonucleotide probe where the second region [D] of the second oligonucleotide probe is a complementarity region which is capable of sequence specific hybridisation to the third oligonucleotide probe to form a double-stranded nucleic acid that is specifically recognised by a nicking agent, and contains the anti-sense sequence of a cleavage site for said nicking agent; the third oligonucleotide probe comprising:
 i. a complementarity region [E] capable of sequence specific hybridisation to the complementarity region [D] in said second oligonucleotide probe to form a double-stranded nucleic acid that is specifically recognised by a nicking agent and contains the cleavage site for said nicking agent; and 
 ii. a second region [F] which is separated from complementarity region [E] following cleavage at said cleavage site to produce a third probe fragment, and which is not capable of sequence specific hybridisation to the complementarity regions [A] and [C] of said first and second oligonucleotide probes; 
   (d) optionally further comprising sequential (3+n)th oligonucleotide probes wherein n is a positive integer, and where the second region of the (2+n)th oligonucleotide probe is a complementarity region which is capable of sequence specific hybridisation to the (3+n)th oligonucleotide probe to form a double-stranded nucleic acid that is specifically recognised by a nicking agent, and contains the anti-sense sequence of a cleavage site for said nicking agent, and the (3+n)th oligonucleotide probe(s) comprise:
 i. a complementarity region capable of sequence specific hybridisation to the complementarity region in said (2+n)th oligonucleotide probe to form a double-stranded nucleic acid that is specifically recognised by a nicking agent and contains the cleavage site for said nicking agent; and 
 ii. a second region which is separated from the first complementarity region described in i. following cleavage at said cleavage site to produce a (3+n)th probe fragment, and which is not capable of sequence specific hybridisation to the complementarity regions of any of said preceding oligonucleotide probes to form a site that is specifically recognised and cleaved by any of the said nicking agents; and 
   (e) one or more nicking agents which specifically recognise the double-stranded nucleic acid that is formed when the complementarity region of the oligonucleotide probes is hybridised to its anti-sense sequence.   
     
     
         60 . A kit according to  claim 59  wherein two or more of the nicking agents are the same. 
     
     
         61 . A kit according to  claim 59  wherein one or more of said oligonucleotide probes is attached to a solid material, or is attached to a moiety that permits attachment of said oligonucleotide probe(s) to a solid material. 
     
     
         62 . A kit according to  claim 59  wherein one or more of the probe fragments produced in (b) and optional (c) and (d) is attached to a moiety that permits its detection, including a colorimetric or fluorometric dye or a moiety that is capable of attachment to a colorimetric dye. 
     
     
         63 . A kit according to  claim 62  wherein said moiety is an enzyme that yields a detectable signal, wherein the detectable signal is a colorimetric or fluorometric signal, following contact with a substrate, including a substrate that is insoluble in water. 
     
     
         64 . A kit according to  claim 59  wherein none of the probe fragments produced in (a), (b), optional (c), and optional (d) are capable of sequence specific hybridisation to the complementarity region of any one of the preceding oligonucleotide probes. 
     
     
         65 . A kit according to  claim 59  wherein one or more of the nicking agents in (a), (b), optional (c), and optional (d) is a naturally occurring enzyme, including a nicking restriction endonuclease. 
     
     
         66 . A kit according to  claim 59  wherein one or more of the nicking agents in (a), (b), optional (c), and optional (d) is an engineered enzyme, including a mutated form of a naturally occurring enzyme, a DNAzyme or a programmable nicking enzyme. 
     
     
         67 . A kit according to  claim 59  wherein one or more of the nicking agents in (a), (b), optional (c), and optional (d) is a double-strand cleaving agent which functions as a nicking agent due to i) strand preference, or ii) only one of the two strands within the double-stranded nucleic acid that is specifically recognised by said double-stranded cleaving agent being capable of cleavage. 
     
     
         68 . A kit according to  claim 59  wherein one or more of the oligonucleotide probes comprises one or more modifications that render it resistant to nuclease, including endonuclease, cleavage. 
     
     
         69 . The kit according to  claim 68  wherein the one or more of the modifications is a phosphorothioate linkage. 
     
     
         70 . A kit according to  claim 59  wherein one or more of the oligonucleotide probes contains multiple copies of the complementarity region. 
     
     
         71 . A kit according to  claim 59  wherein said target nucleic acid is a single-stranded RNA, including single-stranded RNA derived from double-stranded RNA and single-stranded RNA derived from double- stranded DNA, or a single-stranded DNA, including single-stranded DNA derived from double-stranded DNA. 
     
     
         72 . A kit according to  claim 59  wherein the target nucleic acid is viral, or derived from viral nucleic acid material, or is bacterial, or derived from bacterial nucleic acid material. 
     
     
         73 . A kit according to  claim 59  which also comprises one or more of a restriction endonuclease, an exonuclease Ill, a reverse transcriptase, a RNA polymerase or a DNA polymerase. 
     
     
         74 . A kit according to  claim 59  which additionally comprises means to detect the presence of the probe fragment produced from the second oligonucleotide probe and/or one or more of the optional third and (3+n)th oligonucleotide probes. 
     
     
         75 . A kit according to  claim 74  wherein the means to detect the presence of the probe fragment(s) comprises nucleic acid lateral flow. 
     
     
         76 . A kit according to  claim 74  wherein the means to detect the presence of the probe fragment(s) comprises electrical detection. 
     
     
         77 . A kit according  claim 59  for detecting the presence of two or more target nucleic acids of defined sequence which comprises different first, second and optionally third and (3+n)th oligonucleotide probes for each of the two or more target nucleic acids. 
     
     
         78 . A kit according  claim 59  for detecting the presence of two or more target nucleic acids of defined sequence which comprises a different first oligonucleotide probe for each of the two or more different target nucleic acids but the same second and/or optionally third and (3+n)th oligonucleotide probes for each of the two or more target nucleic acids.