METHODS AND COMPOSITIONS FOR DETECTING TARGET NUCLEIC ACIDS

• US 20130005594A1
• Filed: 05/17/2012
• Published: 01/03/2013
• Est. Priority Date: 03/29/2010
• Status: Active Grant

• Current Assignee: Dxterity Diagnostics Incorporated
• Original Assignee: Dxterity Diagnostics Incorporated
• Inventors: Terbrueggen, Robert, Liu, Yenbou, Childers, John Ray Jr., Kim, Chang Hee, Abedi, Majid R.
• Granted Patent: US 10,066,257 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2523/109 chemical ligation between nucleic acids
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

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 ligation 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 ligation product that is directly detected or that is amplified to produce amplicons that are then detected. The present invention also provides methods to stabilize sample RNA so that degradation does not significantly affect the results of the analysis.

Claims

1. A method for detecting a plurality of different target nucleic acids in a sample, wherein each target nucleic acid comprises a first target domain adjacent to a second target domain and a third target capture domain located upstream or downstream from said first and second target domains, said method comprising:

(a) providing a plurality of ligation substrates each comprising;

(i) one of said target nucleic acids;

(ii) a first set of ligation probes comprising;

(A) a first nucleic acid ligation probe comprising;

(1) a first probe domain hybridized to a first target domain of said one target nucleic acid sequence;

(2) a 5′

-ligation moiety; and

(B) a second nucleic acid ligation probe comprising;

(1) a second probe domain hybridized to a second target domain of said one target nucleic acid sequence; and

(2) a 3′

ligation moiety;

(b) ligating said first and second ligation probes without the use of a ligase enzyme to form a first plurality of ligation products;

(c) hybridizing target capture probes comprising a capture moiety to said third target domain of said target nucleic acids to form target complexes;

(d) capturing said target complexes on a surface using said capture moiety;

(e) amplifying said ligation products to form amplicons;

(f) detecting said amplicons,

thereby detecting said target nucleic acids.

2. The method of claim 1, wherein:

said target nucleic acids further comprise a fourth target domain adjacent to a fifth target domain;

said plurality of ligation substrates each further comprises a second set of ligation probes comprising a third nucleic acid ligation probe hybridized to said fourth target domain and a fourth nucleic acid ligation probe hybridized to said fifth target domain;

said method further comprising ligating said third and fourth ligation probes in the absence of a ligase enzyme such that said one target nucleic acid sequence comprises multiple ligation products;

and said detecting step (f) comprises detecting amplicons generated from said multiple ligation products.

3. The method of claim 2, wherein said target nucleic acids further comprise a sixth target domain adjacent to a seventh target domain;

said plurality of ligation substrates each further comprises a third set of ligation probes comprising a fifth nucleic acid ligation probe hybridized to said sixth target domain and a sixth nucleic acid ligation probe hybridized to said seventh target domain;

said method further comprising ligating said third and fourth ligation probes in the absence of a ligase enzyme such that said one target nucleic acid sequence comprises multiple ligation products.

4. The method of claim 1, wherein each of said ligation probes further comprises a primer sequence.

5. The method of claim 1, wherein at least one of said ligation probes of each set of ligation probes further comprise a variable spacer sequence such that said amplicons have a target specific length.

6. The method of claim 1, wherein only one ligation probe of each set of ligation probes comprises said variable spacer sequence.

7. The method of claim 5, wherein said variable spacer sequence is contained between said probe domain and said primer of said ligation probe.

8. The method of claim 1 wherein said capture moiety of said target capture probe comprises a member selected from:

a capture nucleic acid sequence, a bead and a binding partner of a binding partner pair.

9. The method of claim 1, wherein said 5′

ligation moiety comprises DABSYL and said 3′

ligation moiety comprises phosphorothioate.

10. The method of claim 1, wherein said 3′

ligation moiety comprises DABSYL.

11. The method of claim 1, wherein said sample was collected into a buffer comprising guanidinium hydrochloride (GuHCl).

12. The method of claim 11, wherein said sample is blood.

13. The method of claim 1, wherein said target nucleic acids comprise DNA or RNA.

14. The method of claim 1, wherein said detecting step (f) utilizes a technique selected from the group consisting of capillary electrophoresis, mass spectrometry, microarray analysis, sequencing, real-time PCR, optical detection, fluorescence detection, bioluminescence detection, chemiluminescence detection, electrochemical detection, electrochemiluminescence detection and lateral flow detection.

15. The method of claim 1, wherein said hybridizing step (c) occurs simultaneously with said ligating step (b).

21. A method according to claim 1 or 16 wherein said detecting is by capillary electrophoresis.

22. A method according to claim 1 or 16 wherein said detecting is by mass spectrometry.

23. A method according to claim 1 or 16 wherein each of said first primers are the same and each of said second primers are the same.

31. A method for detecting a plurality of target RNA sequences in a sample, wherein each target nucleic acid sequence comprises an adjacent first and second target domain and a third target domain, said method comprising:

(a) collecting a sample into a buffer to form a stabilized sample;

(b) conducting an assay in accordance with claim 1 in said stabilized sample to detect said plurality of target nucleic acids.

32. The method of claim 31, wherein said sample is stable for at least 1 week at ambient room temperature.

16. A method of detecting a plurality of different target nucleic acids in a sample, wherein each target sequence comprises an adjacent first and a second target domain, said method comprising:

a) providing a plurality of ligation substrates each comprising;

i) one of said different target nucleic acids;

ii) a first nucleic acid ligation probe comprising;

1) a first probe domain complementary to a first target domain of said one target nucleic acid;

2) a first primer sequence; and

3) a 5′

-ligation moiety; and

iii) a second nucleic acid ligation probe comprising;

1) a second probe domain complementary to a second target domain of said one target nucleic acid;

2) a second primer sequence; and

3) a 3′

ligation moiety;

wherein one of said ligation probe comprises a variable spacer sequence; and

b) ligating said first and second ligation probes in the absence of a ligase enzyme to form a plurality of different ligation products, wherein different ligation products have different target specific lengths;

c) amplifying said ligation product; and

d) detecting the presence of said different ligation products on the basis of said different target lengths.

17. A method according to claim 16 wherein said target nucleic acid sequences are RNA.

18. A method according to claim 16 wherein said nucleic acid target sequences are DNA.

19. A method according to claim 16, wherein said sample is derived from blood.

20. A method according to claim 16, wherein said sample is derived from paraffin embedded samples.

24. A kit for detecting a target nucleic acid sequence, wherein said target sequence comprises an adjacent first and a second target domain, said kit comprising:

a) 2×

lysis buffer comprising 6 M GuHCl;

b) a first ligation probe comprising;

i) a first probe domain complementary to said first target domain;

ii) a first primer sequence; and

III) a 5′

-ligation moiety; and

c) a second nucleic acid ligation probe comprising;

i) a second probe domain complementary said second target domain;

ii) a second primer sequence; and

iii) a 3′

ligation moiety.

25. A kit according to claim 24 wherein one of said ligation probes further comprises a variable spacer sequence.

26. A method of detecting a plurality of different target nucleic acids in a sample, wherein each target sequence comprises an adjacent first and a second target domain, said method comprising:

a) providing a reaction mixture comprising;

i) a target sample comprising blood; and

ii) 1×

lysis buffer comprising 3 M GuHCl;

b) contacting said reaction mixture with a plurality of different probes sets, each probe set comprising;

i) a first ligation probe comprising;

1) a first probe domain complementary to a first target domain of said one target nucleic acid;

2) a first primer sequence; and

3) a 5′

-ligation moiety; and

ii) a second nucleic acid ligation probe comprising;

1) a second probe domain complementary to a second target domain of said one target nucleic acid;

2) a second primer sequence; and

3) a 3′

ligation moiety;

c) ligating said first and second ligation probes in the absence of a ligase enzyme to form a plurality of different ligation products;

d) amplifying said different ligation products; and

e) detecting the presence of said ligation products.

27. A method according to claim 26 wherein one of said ligation probes further comprises a variable spacer sequence.

28. A method according to claim 26 wherein said target nucleic acids are RNA.

29. A method according to claim 27 wherein one of said first and second ligation probes further comprises a one of a binding partner pair, and prior to said amplifying, a bead comprising the other binding pair is added to capture said ligated products.

30. A method according to claim 27, wherein said detecting is done using said variable spacer sequence.