Nucleic acid detection method

• US 20040002093A1
• Filed: 03/13/2003
• Published: 01/01/2004
• Est. Priority Date: 03/13/2002
• Status: Active Grant

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Ag (China National Chemical Corporation)
• Inventors: Shi, Liang
• Granted Patent: US 7,282,355 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Disclosed are methods for detecting a target nucleic acid molecule having a known sequence among a plurality of nucleic acid molecules. The method includes hybridizing two oligonucleotides to adjacent nucleotide sequences on the target nucleic acid molecule, ligating the two oligonucleotides together, and extending a primer that hybrizes to the 3′ end of the oligonucleotide that hybrized to the 5′ nucleotide sequence on the target nucleic acid molecule. If the target nucleic acid molecule is present (and thereby allowing ligation of the two oligonucleotides), extension of the primer dislodges a detection probe hybridized to the 5′ end of the oligonucleotide that hybridized to the 3′ nucleotide sequence on the target nucleic acid molecule.

Also disclosed are kits for performing the methods of the invention.

Claims

1. A method for detecting the presence of a target nucleic acid molecule in a plurality of templates, comprising:

(a) combining (i) a detection probe comprising a universal sequence;

(ii) a first oligonucleotide comprising a 3′

terminal sequence complementary to a first portion of a known nucleotide sequence of a target nucleic acid molecule and a second sequence complementary to the universal sequence;

(iii) a second oligonucleotide comprising a 5′

terminal sequence complementary to a second portion of the known nucleotide sequence of the target nucleic acid molecule, wherein the second portion of the known nucleotide sequence of the target nucleic acid molecule is adjacent to the first portion of the known nucleotide sequence;

(iv) a primer comprising a 3′

terminal sequence complementary to a portion of the second oligonucleotide, wherein the 3′

terminal sequence is not complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule; and

(v) a plurality of templates suspected of containing the target nucleic acid molecule, wherein the combining is under conditions wherein complementary sequences hybridize to one another;

(b) incubating the product of step (a) with a ligase under conditions wherein the ligase forms a covalent bond between the 3′

terminus of the first oligonucleotide hybridized to the first portion of the known nucleotide sequence and the 5′

terminus of the second oligonucleotide hybridized to the second portion of the known nucleotide sequence;

(c) incubating the product of step (b) with a DNA polymerase under conditions wherein the primer is extended; and

(d) detecting the presence of a non-hybridized detection probe,

wherein the presence of the non-hybridized detection probe indicates the presence of the target nucleic acid molecule in the plurality of templates.

8. The method of claims 1, 2, 3, 4, 5, or 6, wherein the 3′

terminus of the detection probe is labeled.

9. The method of claims 1, 2, 3, 4, 5, or 6, wherein the 5′

terminus of the detection probe is covalently bonded to a quenching moiety.

10. The method of claims 1, 2, 3, 4, 5, or 6, wherein the 5′

terminus of the second oligonucleotide is phosphorylated.

11. The method of claims 1, 2, 3, 4, 5, or 6, wherein the 3′

terminal sequence of the first oligonucleotide complementary to the first portion of the known nucleotide sequence of the target nucleic acid molecule is about twenty nucleotides in length.

12. The method of claims 1, 2, 3, 4, 5, or 6, wherein the 5′

terminal sequence of the second oligonucleotide complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule is about twenty nucleotides in length.

13. The method of claims 1, 2, 3, 4, 5, or 6, wherein the universal sequence is about twenty nucleotides in length.

14. The method of claims 1, 2, 3, 4, 5, or 6, wherein the first oligonucleotide is about sixty nucleotides in length. 15 The method of claims 1, 2, 3, 4, 5, or 6, wherein the second oligonucleotide is about forty nucleotides in length.

16. The method of claims 1, 2, 3, 4, 5, or 6, wherein the 3′

terminal sequence of the primer is about twenty nucleotides in length.

18. The method of claims 1, 2, 3, 4, 5, or 6, wherein the non-hybridized detection probe is shortened and wherein the presence of the shortened non-hybridized detection probe indicates the presence of the target nucleic acid molecule in the plurality of templates.

2. A method for detecting the presence of a target nucleic acid molecule in a plurality of templates, comprising:

(a) combining (i) a detection probe comprising a universal sequence;

(ii) a first oligonucleotide comprising a 3′

terminal sequence complementary to a first portion of a known nucleotide sequence of a target nucleic acid molecule and a second sequence complementary to the universal sequence;

(iii) a second oligonucleotide comprising a 5′

terminal sequence complementary to a second portion of the known nucleotide sequence of the target nucleic acid molecule, wherein the second portion of the known nucleotide sequence of the target nucleic acid molecule is adjacent to the first portion of the known nucleotide sequence;

(iv) a primer comprising a 3′

terminal sequence complementary to a portion of the second oligonucleotide, wherein the 3′

terminal sequence is not complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule; and

(v) a plurality of templates suspected of containing the target nucleic acid molecule;

wherein the combining is under conditions wherein complementary sequences hybridize to one another;

(b) incubating the product of step (a) with a ligase under conditions wherein the ligase forms a covalent bond between the 3′

terminus of the first oligonucleotide hybridized to first portion of the known nucleotide sequence and the 5′

terminus of the second oligonucleotide hybridized to the second portion of the known nucleotide sequence;

(c) incubating the product of step (b) with a DNA polymerase and a second primer, wherein the 3′

terminal sequence of the second primer is identical to a portion of the first oligonucleotide that is 5′

to both the 3′

terminal sequence and the second sequence of the first oligonucleotide, under conditions wherein the target nucleic acid molecule is amplified by extension of the primer and second primer; and

(d) detecting the presence of a non-hybridized detection probe,

wherein the presence of the non-hybridized detection probe indicates the presence of the target nucleic acid molecule in the plurality of templates.

7. The method of claims 2, 4, or 6, wherein the nucleotide sequence of the primer and the second primer is the same.

17. The method of claims 2, 4, or 6, wherein the 3′

terminal sequence of the second primer is about twenty nucleotides in length.

3. A method for detecting the presence of a target nucleic acid molecule in a plurality of templates, comprising:

(a) combining (i) a detection probe comprising a universal sequence;

(ii) a first oligonucleotide comprising a 3′

terminal sequence complementary to a first portion of a known nucleotide sequence of a target nucleic acid molecule and a second sequence complementary to the universal sequence;

(iii) a second oligonucleotide comprising a 5′

terminal sequence complementary to a second portion of the known nucleotide sequence of the target nucleic acid molecule, wherein the second portion of the known nucleotide sequence of the target nucleic acid molecule is adjacent to the first portion of the known nucleotide sequence; and

(iv) a plurality of templates suspected of containing the target nucleic acid molecule;

wherein the combining is under conditions wherein complementary sequences hybridize to one another;

(b) incubating the product of step (a) with a ligase under conditions wherein the ligase forms a covalent bond between the 3′

terminus of the first oligonucleotide hybridized to first portion of the known nucleotide sequence and the 5′

terminus of the second oligonucleotide hybridized to the second portion of the known nucleotide sequence;

(c) incubating the product of step (b) with a DNA polymerase and a primer comprising a 3′

terminal sequence complementary to a portion of the second oligonucleotide, wherein the 3′

terminal sequence is not complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule, under conditions wherein the primer is extended; and

(d) detecting the presence of a non-hybridized detection probe,

wherein the presence of the non-hybridized detection probe indicates the presence of the target nucleic acid molecule in the plurality of templates.

4. A method for detecting the presence of a target nucleic acid molecule in a plurality of templates, comprising:

(a) combining (i) a detection probe comprising a universal sequence;

(ii) a first oligonucleotide comprising a 3′

terminal sequence complementary to a first portion of a known nucleotide sequence of a target nucleic acid molecule and a second sequence complementary to the universal sequence;

(iii) a second oligonucleotide comprising a 5′

terminal sequence complementary to a second portion of the known nucleotide sequence of the target nucleic acid molecule, wherein the second portion of the known nucleotide sequence of the target nucleic acid molecule is adjacent to the first portion of the known nucleotide sequence; and

(iv) a plurality of templates suspected of containing the target nucleic acid molecule;

wherein the combining is under conditions wherein complementary sequences hybridize to one another;

(b) incubating the product of step (a) with a ligase under conditions wherein the ligase forms a covalent bond between the 3′

terminus of the first oligonucleotide hybridized to first portion of the known nucleotide sequence and the 5′

terminus of the second oligonucleotide hybridized to the second portion of the known nucleotide sequence;

(c) incubating the product of step (b) with a DNA polymerase, a primer comprising a 3′

terminal sequence complementary to a portion of the second oligonucleotide, wherein the 3′

terminal sequence is not complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule, and a second primer, wherein the 3′

terminal sequence of the second primer is identical to a portion of the first oligonucleotide that is 5′

to both the 3′

terminal sequence and the second sequence of the first oligonucleotide, under conditions wherein the target nucleic acid molecule is amplified by extension of the primer and second primer; and

(d) detecting the presence of a non-hybridized detection probe,

wherein the presence of the non-hybridized detection probe indicates the presence of the target nucleic acid molecule in the plurality of templates.

5. A method for detecting the presence of a target nucleic acid molecule in a plurality of templates, comprising:

(a) combining (i) a first oligonucleotide comprising a 3′

terminal sequence complementary to a first portion of a known nucleotide sequence of a target nucleic acid molecule and a second sequence complementary to a universal sequence;

(ii) a second oligonucleotide comprising a 5′

terminal sequence complementary to a second portion of the known nucleotide sequence of the target nucleic acid molecule, wherein the second portion of the known nucleotide sequence of the target nucleic acid molecule is adjacent to the first portion of the known nucleotide sequence; and

(iii) a plurality of templates suspected of containing the target nucleic acid molecule;

wherein the combining is under conditions wherein complementary sequences hybridize to one another;

(b) incubating the product of step (a) with a detection probe comprising a universal sequence under conditions where the detection probe hybridizes to the second sequence of the first oligonucleotide and a ligase under conditions wherein the ligase forms a covalent bond between the 3′

terminus of the first oligonucleotide hybridized to first portion of the known nucleotide sequence and the 5′

terminus of the second oligonucleotide hybridized to the second portion of the known nucleotide sequence;

(c) incubating the product of step (b) with a DNA polymerase and a primer comprising a 3′

terminal sequence complementary to a portion of the second oligonucleotide, wherein the 3′

terminal sequence is not complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule, under conditions wherein the primer is extended; and

(d) detecting the presence of a non-hybridized detection probe,

wherein the presence of the non-hybridized detection probe indicates the presence of the target nucleic acid molecule in the plurality of templates.

6. A method for detecting the presence of a target nucleic acid molecule in a plurality of templates, comprising:

(a) combining (i) a first oligonucleotide comprising a 3′

terminal sequence complementary to a first portion of a known nucleotide sequence of a target nucleic acid molecule and a second sequence complementary to a universal sequence;

(ii) a second oligonucleotide comprising a 5′

terminal sequence complementary to a second portion of the known nucleotide sequence of the target nucleic acid molecule, wherein the second portion of the known nucleotide sequence of the target nucleic acid molecule is adjacent to the first portion of the known nucleotide sequence; and

(iii) a plurality of templates suspected of containing the target nucleic acid molecule;

wherein the combining is under conditions wherein complementary sequences hybridize to one another;

(b) incubating the product of step (a) with a detection probe comprising a universal sequence under conditions where the detection probe hybridizes to the second sequence of the first oligonucleotide and a ligase under conditions wherein the ligase forms a covalent bond between the 3′

terminus of the first oligonucleotide hybridized to first portion of the known nucleotide sequence and the 5′

terminus of the second oligonucleotide hybridized to the second portion of the known nucleotide sequence;

(c) incubating the product of step (b) with a DNA polymerase, a primer comprising a 3′

terminal sequence complementary to a portion of the second oligonucleotide, wherein the 3′

terminal sequence is not complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule, and a second primer, wherein the 3′

terminal sequence of the second primer is identical to a portion of the first oligonucleotide that is 5′

to both the 3′

terminal sequence and the second sequence of the first oligonucleotide, under conditions wherein the target nucleic acid molecule is amplified by extension of the primer and second primer; and

(d) detecting the presence of a non-hybridized detection probe,

wherein the presence of the non-hybridized detection probe indicates the presence of the target nucleic acid molecule in the plurality of templates.

19. A kit for detecting the presence of a target nucleic acid molecule in a plurality of templates, comprising:

(i) a detection probe comprising a universal sequence;

(ii) a first oligonucleotide comprising a 3′

terminal sequence complementary to a first portion of a known nucleotide sequence of a target nucleic acid molecule and a second sequence complementary to the universal sequence;

(iii) a second oligonucleotide comprising a 5′

terminal sequence complementary to a second portion of the known nucleotide sequence of the target nucleic acid molecule, wherein the second portion of the known nucleotide sequence of the target nucleic acid molecule is adjacent to the first portion of the known nucleotide sequence; and

(iv) a primer comprising a 3′

terminal sequence complementary to a portion of the second oligonucleotide, wherein the 3′

terminal sequence is not complementary to the second portion of the known nucleotide sequence of the target nucleic acid molecule; and

(v) means for detecting a non-hybridized detection probe.

20. The kit of claim 19 further comprising a ligase.

21. The kit of claim 19 further comprising a DNA polymerase.

22. The kit of claim 19 further comprising a second primer, wherein the 3′

terminal sequence of the second primer is identical to a portion of the first oligonucleotide that is 5′

to both the 3′

terminal sequence and the second sequence of the first oligonucleotide.

23. The kit of claim 19, wherein the 5′

terminal sequence of the first oligonucleotide is complementary to the 3′

sequence of the second oligonucleotide.

24. The kit of claim 19, wherein the means for detecting a non-hybridized detection probe is able to detect a shortened non-hybridized detection probe.