Chemical ligation dependent probe amplification (CLPA)
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 one aspect, the ligation product is of variable length that correlates with a particular target. Following chemical ligation, the probes may be amplified and detected by capillary electrophoresis or microarray analysis.
1. A method for detecting in a sample, comprising a plurality of sample nucleic acids of different nucleic acid sequences, the presence of at least one specific target nucleic acid sequence comprising a first and a second target domain, the domains located adjacent to one another, comprising the steps of:
(a) contacting the sample nucleic acids with a plurality of different probes sets, each probe set comprising:
(i) a first ligation probe comprising:
(1) a first probe domain complementary to said first target domain;
(2) a first non-complementary region being non-complementary to the said target nucleic acid sequence; and
(3) a 5′-ligation moiety comprising a DABSYL moiety; and
(ii) second ligation probe comprising:
(1) a second probe domain complementary to said second target domain;
(2) a second non-complementary region, being non-complementary to the said target nucleic acid sequence;
(3) a 3′ ligation moiety comprising a phosphorothioate moiety;
(b) ligating said first and second ligation probes in the absence of a ligase enzyme to form a ligation product; wherein at least one of said ligation probes comprises a variable spacer nucleic acid sequence such that each ligation product is a different length;
(c) amplifying said ligation product to form ligation amplicons under conditions whereby a fluorescent label is incorporated into said amplicons; and
(d) detecting the presence of said ligation amplicons by detecting the presence of said fluorescent label.
2. A method of claim 1 , wherein said target nucleic acid sequence is RNA and/or DNA.
3. A method of claim 1 , wherein said target nucleic acid sequence comprises unpurified RNA.
4. A method as in claim 1 , wherein said detecting step is by capillary electrophoresis.
5. A method for detecting in a sample, comprising a plurality of sample nucleic acids of different nucleic acid sequences, the presence of at least one specific target nucleic acid sequence comprising a first and a second target domain, the domains located adjacent to one another, comprising the steps of:
(a) contacting the sample nucleic acids with a plurality of different probes sets, each probe set comprising:
(i) a first ligation probe comprising:
(1) a first probe domain complementary to said first target domain;
(2) a first non-complementary region being non-complementary to the said target nucleic acid sequence; and
(3) a 5′-ligation moiety comprising a halogen leaving group; and
(ii) second ligation probe comprising:
(1) a second probe domain complementary to said second target domain;
(2) a second non-complementary region, being non-complementary to the said target nucleic acid sequence;
(3) a 3′ ligation moiety comprising a phosphorothioate moiety;
(b) ligating said first and second ligation probes in the absence of a ligase enzyme to form a ligation product; wherein at least one of said ligation probes comprises a variable spacer nucleic acid sequence such that each ligation product is a different length;
(c) amplifying said ligation product to form ligation amplicons under conditions whereby a fluorescent label is incorporated into said amplicons; and
(d) detecting the presence of said ligation amplicons by detecting the presence of said fluorescent label.
6. A method of claim 5 , wherein said target nucleic acid sequence is RNA and/or DNA.
7. A method of claim 5 , wherein said target nucleic acid sequence comprises unpurified RNA.
8. A method as in claim 5 , wherein said detecting step is by capillary electrophoresis.
9. A method for detecting in a sample, comprising a plurality of sample nucleic acids of different nucleic acid sequences, the presence of at least one specific target nucleic acid sequence comprising a first and a second target domain, the domains located adjacent to one another, comprising the steps of:
(a) contacting the sample nucleic acids with a plurality of different probes sets, each probe set comprising:
(i) a first ligation probe comprising:
(1) a first probe domain complementary to said first target domain;
(2) a first non-complementary region being non-complementary to the said target nucleic acid sequence; and
(3) a 5′-ligation moiety comprising a leaving group; and
(ii) second ligation probe comprising:
(1) a second probe domain complementary to said second target domain;
(2) a second non-complementary region, being non-complementary to the said target nucleic acid sequence;
(3) a 3′ ligation moiety comprising a nucleophilic group;
(b) ligating said first and second ligation probes in the absence of a ligase enzyme to form a ligation product; wherein at least one of said ligation probes comprises a variable spacer nucleic acid sequence such that each ligation product is a different length;
(c) amplifying said ligation product to form ligation amplicons under conditions whereby a fluorescent label is incorporated into said amplicons; and
(d) detecting the presence of said ligation amplicons by detecting the presence of said fluorescent label.
10. A method of claim 9 , wherein said target nucleic acid sequence is RNA and/or DNA.
11. A method of claim 9 , wherein said target nucleic acid sequence comprises unpurified RNA.
12. A method as in claim 9 , wherein said detecting step is by capillary electrophoresis.