Detection of nucleic acid targets using chemically reactive oligonucleotide probes
The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more oligonucleotide probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligated oligonucleotide product. In accordance with the invention, the presence of the target(s) of interest can be determined by measuring the presence or amount of ligated oligonucleotide product.
1. A method for detecting the presence of a known target nucleic acid in a sample comprising:
a) providing a ligation substrate comprising:
i) said known target nucleic acid comprising a first target domain and an adjacent second target domain, wherein the first target domain is upstream of the second target domain;
ii) a first nucleic acid ligation probe comprising:
1) a first probe domain hybridized to said first target domain; and
2) a 5′-ligation moiety; and
iii) a second nucleic acid ligation probe comprising:
1) a second probe domain hybridized to said second target domain;
2) a 3′ ligation moiety;
wherein at least one of said first and said second ligation probes comprises an anchor sequence;
b) ligating said first and said second ligation probes in the absence of a ligase enzyme to form a ligation product;
c) capturing said ligation product on a substrate comprising a capture probe that hybridizes to said anchor sequence; and
d) detecting the presence of said ligation product thereby detecting the presence of said target nucleic acid.
2. A method according to claim 1 wherein said first and second ligation moieties form a ligation moiety pair selected from the group consisting of a) a thioester and a nucleophile and b) a dimethylamino(azobenzene) sulfonyl (DABSYL) moiety and a phosphothioate moiety.
3. A method according to claim 1 wherein said sample is selected from the group consisting of a mammalian bodily fluid and a mammalian paraffin embedded tissue sample.
4. A method according to claim 1 wherein the other of said first and second ligation probes comprises a detectable label.
5. A method according to claim 4 wherein said detectable label is a fluorophore.
6. A method according to claim 1 wherein the other of said first and second ligation probes comprises a label binding sequence, and said ligated product further comprises a label probe hybridized to said binding sequence, wherein said label probe comprises a detectable label.
7. A method according to claim 1 wherein said target nucleic acid is DNA.
8. A method according to claim 1 wherein said known target nucleic acid is RNA.
9. A method for detecting the presence of a known target nucleic acid in a sample comprising:
a) providing a ligation substrate comprising:
i) said known target nucleic acid comprising a first target domain and an adjacent second target domain, wherein the first target domain is upstream of the second target domain;
ii) a first nucleic acid ligation probe comprising:
1) a first probe domain hybridized to said first target domain; and
2) a 5′-ligation moiety; and
iii) a second nucleic acid ligation probe comprising:
1) a second probe domain hybridized to said second target domain;
2) a 3′ ligation moiety;
wherein said first and second ligation moieties form a ligation moiety pair selected from the group consisting of a) a thioester and a nucleophile and b) a dimethylamini(azobenzene) sulfonyl (DABSYL) moiety and a phosphothioate moiety, and wherein at least one of said first and said second nucleic acid ligation probes comprises an anchor sequence;
b) ligating said first and said second ligation probes in the absence of a ligase enzyme to form a ligation product;
c) capturing said ligation product on a substrate comprising a capture probe complementary to said anchor sequence; and
d) detecting the presence of said ligation product.
10. A method for detecting the presence of a known target nucleic acid in a sample, wherein said sample is selected from the group consisting of a mammalian bodily fluid and a mammalian paraffin embedded tissue sample, said method comprising:
a) providing a ligation substrate comprising:
i) said known target nucleic acid comprising a first target domain and an adjacent second target domain, wherein the first target domain is upstream of the second target domain;
ii) a first nucleic acid ligation probe comprising:
1) a first probe domain hybridized to said first target domain; and
2) a 5′-ligation moiety; and
iii) a second nucleic acid ligation probe comprising:
1) a second probe domain hybridized to said second target domain;
2) a 3′ ligation moiety;
wherein at least one of said first and said second nucleic acid ligation probes comprises an anchor sequence;
b) ligating said first and said second ligation probes in the absence of a ligase enzyme to form a ligation product;
c) capturing said ligation product on a substrate comprising a capture probe complementary to said anchor sequence; and
d) detecting the presence of said ligation product.