Method and kit for nucleic acid sequence detection

• US 20070269825A1
• Filed: 03/08/2007
• Published: 11/22/2007
• Est. Priority Date: 03/08/2006
• Status: Active Grant

• Current Assignee: Atila Biosystems, Inc.
• Original Assignee: Atila Biosystems, Inc.
• Inventors: Wang, Youxiang, Tao, Wenjing
• Granted Patent: US 8,673,567 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

A method and kit for detecting the presence of a target sequence in a polynucleotide analyte contained in a sample are disclosed. In practicing the method, the sample is mixed with a target probe having a sequence capable of hybridizing with said target sequence, under conditions effective to form a double-stranded complex of the analyte and the probe, and the probe in the complex is reacted in the presence of a polymerase and a selected one to three of four possible nucleotide triphosphates, to add a selected one or more target-directed nucleotide bases to the probe'"'"'s 3′ end to produce a modified probe. The modified probe is hybridized with a detection probe, the two probes are ligated to form a two-probe ligation product, and the presence of the ligation product is detected.

Claims

1. A method of detecting the presence of a target sequence in a polynucleotide analyte contained in a sample, comprising:

(a) mixing the sample with a target probe having a sequence capable of hybridizing with said target sequence, under conditions effective to form a double-stranded complex of the analyte and the probe,

(b) reacting the probe in the complex in the presence of a polymerase and a selected one to three of four possible nucleotide triphosphates, thereby to add a selected one or more target-directed nucleotide bases to the probe'"'"'s 3′

end to produce a modified probe,

(c) hybridizing the modified probe with a detection probe, where the detection probe does not hybridize to the target and at least one of the two probes has a sticky end having a single-stranded portion complementary to the other probe'"'"'s single-stranded portion at its 3′

end or 5′

end.

(d) by said reacting ligating the two probes to form a two-probe ligation product, and

(e) detecting the presence of the ligation product.

2. The method of claim 1, wherein step (b) includes the additional step to dissociate the modified probe from the target by denaturation or degrading the target.

3. The method of claim 1, wherein the target probe has a sticky end before or after polymerization reaction in step (b), and the sticky end'"'"'s single-stranded portion contains one or more than one nucleotides.

4. The method of claim 1, both detection probe and modified probe are single-stranded polynucleotides having sticky ends complementary to each other in step (3), and said two probe ligation product formed in step (d) is a circular single-stranded polynucleotides.

5. The method of claim 4, wherein step (d) includes the additional step of digesting any linear probes and target after ligation reaction.

6. The method of claim 1, wherein the amount of target probe in step (a) is in substantial molar excess of the amount of target analyte, and which further includes repeating steps (a) and (b) to increase the modified probes present in the sample, and said repeating steps (a) and (b) includes heating the sample after each step (b) to release modified probe from the target polynucleotide, and cooling the sample as part of each step (a) to hybridize unreacted target probe with said target sequence.

7. The method of claim 1, for use in detecting a 1-6 base target region of interest, such as a SNP or known-location point mutation, wherein said target probe has a 3′

-end nucleotide base that is partially complementary to or immediately adjacent the target region of interest, and wherein step (b) is carried out so the 3′

-end sequence of the modified probe is complementary to the target region of interest.

8. The method of claim 7, for use in detecting a plurality of target sequences of interest in one or more polynucleotides in a sample, wherein the sample is mixed with a plurality of target probes, each is a single-stranded polynucleotides having a hairpin loop structure capable of forming a double-stranded complex with a region of a sample polynucleotide, wherein step (a) includes dividing the sample into two or more sample aliquots and mixing each sample aliquot with the plurality of target probes, step (b) includes reacting each sample aliquot with a selected one to three of four different nucleotide triphosphates in the presence of polymerase, a different combination of one to three of four nucleotide triphosphate for each aliquot, step (c) includes hybridizing the modified probe with a aliquot-specific detection probe, step (d) includes reacting ligating the modified probe with an aliquot-specific detection probe to form ligation products, and step (e) includes amplification of the ligation products in each aliquot before detection.

9. The method of claim 8, wherein step (e) includes the additional step of mixing each aliquot prior to detection by hybridizing with probes attached on solid surfaces.

10. The method of claim 8, wherein said the plurality of target probes are synthesized from solid surface in a reaction vessel, and releasing the plurality of target probes from solid surfaces forms a mixture of plurality of target probes.

11. The method of claim 8, wherein step (e) includes additional step of dividing ligation products in each aliquot into two or more aliquots and amplifying each aliquot with specific primers before detection.

12. The method of claim 1, wherein said detecting the presence of the ligation product comprises using the ligation product as a template for amplification to produce detectable amplicons.

13. The method of claim 1, wherein the one of the target or detection probes is attached to a solid support, the ligation product produced in step (d) is also attached to the solid support, and and step (e) of detecting the presence of the ligation product includes detecting the presence of the ligation product on the support.

14. The method of claim 1, for use in protein-binding assay, wherein the polynucleotide analyte is carried on a protein-binding agent.

15. The method of claim 14, for use in detecting a plurality of polynucleotide analytes in a sample for protein-binding assay, wherein the sample is incubated with a plurality of protein-binding agents to form a plurality of protein-binding polynucleotides analytes, wherein step (a) includes mixing plurality of protein-binding polynucleotides analytes with a plurality of target probes to generate the plurality of hybridizing complexes, step (b) reacting the hybridizing complexes with a selected one to three of four different nucleotide triphosphates in the presence of a polymerase to produce modified probes, step (c) includes hybridizing the modified probes with detection probes, step (d) includes reacting ligating the modified probes with detection probes to form ligation products, step (e) includes dividing ligation products into two or more aliquots and amplifying each aliquot with specific primers to produce amplicons and detection each amplicons.

16. The method of claim 15, wherein detection ligation products produced in step (d) includes amplifying the ligation products to produce amplicons and then hybridizing the amplicons with probes attached on solid surface.

17. The method of claim 1, wherein said target polynucleotide is an RNA polynucleotide and the polymerase is a reverse transcriptase polymerase. reaction steps (a)-(e) are carried out by addition of the components required in the steps to a single reaction vessel, and carrying out the steps under substantially isothermal conditions.

18. The method of claim 17, wherein steps (b) includes degrading RNA analyte after or during forming modified probes.

19. The method of claim 17, wherein the amount of target probe in step (a) is in substantial molar excess of the amount of target analyte, and which further includes repeating steps (a) and (b) to increase the modified probes present in the sample, wherein repeating steps (a) and (b) includes heating the sample after each step (b) to release modified probe from the target polynucleotide, and cooling the sample as part of each step (a) to hybridize unreacted target probe with said target sequence.

20. The method of claim 17, wherein said detection probe includes a promoter, the ligation product produced in step (d) containing the promoter sequences, and step (e) includes reacting the two-probe ligation product with a promoter-dependent polymerase under conditions effective to promote synthesis of transcripts containing the target-probe sequence, and detecting the presence of the transcripts.

21. The method of claim 20, wherein detecting the presence of the transcripts in step (e) includes reacting the transcripts with molecular beacon probes contained in the reaction medium used to generate the transcripts.

22. The method of claim 17, which further includes repeating steps (a)-(e), where the target sequence in step (a) is supplied by said transcripts.

23. A kit for detection of an RNA analyte, such as an RNA viral genome, an siRNA, or an miRNA, having a known target sequence, comprising,

(a) a single-stranded DNA target probe having a sequence capable of hybridizing with said RNA target sequence, under conditions effective to form a double-stranded heteroduplex complex of the analyte and the probe,

(b) a reverse transcriptase enzyme,

(c) one to three of four possible nucleotide triphosphates,

(d) a single-stranded DNA detection probe with a sticky end having a single strand portion that is complementary to a 3′

-end sequence of the target probe after addition of the selected nucleotide(s) to the 3′

-end of the target probe, where the detection probe (i) does not hybridize to the target and (ii) contains a promoter sequences,

(e) a polymerase, and

(f) a detection label capable of producing a detectable signal thereby,

wherein addition of an RNA analyte and aqueous medium to a reaction vessel, and addition of the kit components, is effective to initiate reactions to (a) form a double-stranded complex of the analyte and target probe, (b) add a selected one or more target-directed nucleotide bases to the target probe'"'"'s 3′

end and a reverse transcriptase to produce modified probe, (c) ligate the modified target probe and the detection probe to form a two-probe ligation product, (d) amplifying ligation-product to produce amplicons containing analyte-target sequences, and (e) bind the detection label to said amplicons, to produce a detectable signal in the vessel.

24. The kit of claim 23, for use in detecting a plurality of target sequences of interest in one or more RNA analytes, wherein a plurality of single stranded target probes, each having a hairpin loop structure are capable of forming a hybridizing complex with the plurality of target sequences.

25. The kit of claim 23, wherein said the promoter sequences in the detection probe is a RNA polymerase promoter, and said polymerase is RNA polymerase.