Method for detecting a target nucleic acid molecule

• US 9,771,612 B2
• Filed: 09/15/2014
• Issued: 09/26/2017
• Est. Priority Date: 03/21/2012
• Status: Active Grant

• Current Assignee: Olympus Corporation
• Original Assignee: Olympus Corporation
• Inventors: Hori, Kunio, Fujimoto, Kenzo
• Primary Examiner(s): Lu, Frank
• Application Number:
  US14/486,030
• Publication Number:
  US 20150004607A1
• Time in Patent Office: 1,107 Days
• Field of Search: 435 61, 435 611, 435 911, 435183, 436 94, 436501, 536 231, 536 243, 536 2433
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2549/10 the purpose being that of reducing false positive or false negative signals
• CPC Class Codes: C12Q 2563/107 fluorescence
• 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 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

A method for detecting a target nucleic acid comprising: forming a three-component association product by allowing the association of at least a nucleic acid molecule, a first nucleic acid probe having a first marker bound thereto, and a second nucleic acid probe having a second marker bound thereto; forming at least one covalent bond between the target nucleic acid molecule and the first nucleic acid probe and between the target nucleic acid molecule and the second nucleic acid probe; and binding the three-component association product to a solid phase carrier through the second marker; recovering the three-component association product bound to the solid phase carrier; releasing the first marker from the recovered three-component association product; and detecting the target nucleic acid molecule by detecting the free first marker.

Claims

1. A method for detecting a target nucleic acid molecule in a nucleic acid-containing sample, comprising:

(a) preparing a sample solution by adding said nucleic acid-containing sample, a first nucleic acid probe that has a first marker bound thereto and specifically hybridizes with a region of the target nucleic acid molecule, and a second nucleic acid probe that has a second marker bound thereto and specifically hybridizes with a region of the target nucleic acid molecule that differs from the region hybridized by the first nucleic acid probe, wherein the first marker is a luminescent substance;

(b) denaturing the target nucleic acid molecule in the sample solution prepared in step (a);

(c) allowing the target nucleic acid molecule, the first nucleic acid probe, and the second nucleic acid probe in the sample solution to associate each other after step (b);

(d) forming at least one covalent bond between the target nucleic acid molecule and the first nucleic acid probe and forming at least one covalent bond between the target nucleic acid molecule and the second nucleic acid probe, thereby forming a three-component association product comprising the target nucleic acid molecule, the first nucleic acid probe and the second nucleic acid probe;

(e) adding, after step (d), a solid phase carrier with a site that binds with the second marker to the sample solution such that the three-component association product binds to the solid phase carrier through the second marker in the three-component association product, recovering the three-component association product bound to the solid phase carrier by a solid-liquid separation procedure and forming a recovered three-component association product;

(f) releasing the first marker from the recovered three-component association product after step (e); and

(g) detecting the target nucleic acid molecule by detecting the first marker released from the recovered three-component association product after step (f).

2. The method according to claim 1, wherein

in step (f), the first marker is released from the three-component association product by:

(f1) irradiating the three-component association product with an ultraviolet light at 300 nm to 380 nm under conditions in which the target nucleic acid molecule, the first nucleic acid probe and the second nucleic acid probe of the three-component association product dissociate each other if a covalent bond is not formed between the target nucleic acid molecule and the first nucleic acid probe or between the target nucleic acid molecule and the second nucleic acid probe;

(f2) decomposing the three-component association product with a nucleolytic enzyme;

(f3) heating the three-component association product to 80°

C. or higher which is a temperature sufficiently higher than the melting temperature (T_m) value of the first nucleic acid probe;

or,

(f4) heating the three-component association product to 50°

C. to 100°

C. under alkaline conditions.

3. The method according to claim 2, wherein step (f1) is carried out in a solution containing a salt at a concentration at which the T_m value of the first nucleic acid probe is 25°

C. or lower.

4. The method according to claim 1, wherein each of the first nucleic acid probe and the second nucleic acid probe comprise a photoreactive base derivative, and

wherein the covalent bond between the target nucleic acid molecule and the first nucleic acid probe and the covalent bond between the target nucleic acid molecule and the second nucleic acid probe are formed by a photochemical reaction mediated by the photoreactive base derivative.

5. The method according to claim 4, wherein at least one base in a region of the first nucleic acid probe that hybridizes with the target nucleic acid molecule is substituted with a photoreactive base derivative, and at least one base in a region of the second nucleic acid probe that hybridizes with the target nucleic acid molecule is substituted with a photoreactive base derivative.

6. The method according to claim 4, wherein the photoreactive base derivative is 3-cyanovinylcarbazole nucleoside, and the covalent bond between the target nucleic acid molecule and the first nucleic acid probe and the covalent bond between the target nucleic acid molecule and the second nucleic acid probe are formed by irradiating the sample solution with a light at 340 nm to 380 nm in step d).

7. The method according to claim 1, wherein,

prior to step (f),

the three-component association product bound to the solid phase carrier recovered in step (e) is washed with a washing solution having a salt concentration at which the Tm value of the first nucleic acid probe is 25°

C. or lower.

8. The method according to claim 1,

wherein the luminescent substance of the first nucleic acid probe produces a fluorescent signal, and

wherein, in step (g), said detecting the first marker released from the recovered three-component association product is carried out using a fluorescent single molecule measurement method.

9. The method according to claim 8, wherein the fluorescent single molecule measurement method comprises:

(h) calculating the number of molecules of the first marker released from the recovered three-component association product using fluorescence correlation spectroscopy or fluorescence intensity distribution analysis, or

(i) calculating the number of molecules of the first marker released from the recovered three-component association product using a confocal microscope or multi-photon microscope.

10. A method for detecting a target nucleic acid molecule in a nucleic acid-containing sample, comprising:

(a) preparing a sample solution obtained by adding said nucleic acid-containing sample, a first nucleic acid probe that has a first marker bound thereto and specifically hybridizes with a region of the target nucleic acid molecule, a second nucleic acid probe that has a second marker bound thereto and specifically hybridizes with a region of the target nucleic acid molecule that differs from the region hybridized by the first nucleic acid probe, and a solid phase carrier with a site that binds with the second marker, wherein the first marker is a luminescent substance;

(b) denaturing the target nucleic acid molecule in the sample solution prepared in step (a);

(c) allowing the target nucleic acid molecule, the first nucleic acid probe, and the second nucleic acid probe in the sample solution to associate each other after step (b);

(d) forming at least one covalent bond between the target nucleic acid molecule and the first nucleic acid probe and forming at least one covalent bond between the target nucleic acid molecule and the second nucleic acid probe, thereby forming a three-component association product bound to the solid phase carrier comprising the target nucleic acid molecule, the first nucleic acid probe and the second nucleic acid probe;

(e) recovering the three-component association product bound to the solid phase carrier by solid-liquid separation procedure and forming a recovered three-component association product;

(f) releasing the first marker from the recovered three-component association product after step (e); and

(g) detecting the target nucleic acid molecule by detecting the first marker released from the recovered three-component association product after step (f).

11. The method according to claim 10, wherein, in step (a), the second nucleic acid probe is bound to the solid phase carrier through the second marker.