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Method for detecting target nucleic acid with specific base sequence and set of nucleic acids for detection

Foreign code F110005554
File No. N081-31WO
Posted date Sep 7, 2011
Country United States of America
Application number 08510106
Gazette No. 20090221429
Gazette No. 7851159
Date of filing Nov 17, 2006
Gazette Date Sep 3, 2009
Gazette Date Dec 14, 2010
International application number JP2006323031
International publication number WO2007058326
Date of international filing Nov 17, 2006
Date of international publication May 24, 2007
Priority data
  • P2005-332424 (Nov 17, 2005) JP
  • 2006WO-JP323031 (Nov 17, 2006) WO
Title Method for detecting target nucleic acid with specific base sequence and set of nucleic acids for detection
Abstract (US7851159)
It is intended to provide a method for detecting a target nucleic acid with a specific base sequence existing in a sample mixture with high specificity and sensitivity by utilizing the formation of a hybrid with a complementary strand as a detection principle, and a nucleic acid for the detection.
The invention relates to a set of nucleic acids for detecting a target nucleic acid with a specific base sequence comprising a photoligating nucleic acid composed of a nucleic acid (with the proviso that in the nucleic acid, a nucleic acid and a peptide nucleic acid are included) having a group represented by the formula I, II, III, IV or V described in claims at the 5′ end or 3′ end as a base moiety, and a photoligated nucleic acid having a base with a carbon-carbon double bond at the 3′ end or 5′ end as a base moiety capable of photoligating to the photoligating nucleic acid, wherein either of the photoligating nucleic acid and the photoligated nucleic acid has a labeling moiety and the other remaining nucleic acid is immobilized on a substrate, and the method for detecting a target nucleic acid with a specific base sequence by using the set of nucleic acids.
Scope of claims [claim1]
1. A method for detecting a target nucleic acid having a specific base sequence contained in a nucleic acid mixture, wherein: utilizing a set of nucleic acids for detecting a target nucleic acid having a specific base sequence, which the set of nucleic acids includes a photoligating nucleic acid comprising a nucleic acid having a group represehted by the following Formula I, Formula II, Formula III, Formula IV or Formula V:

wherein Z represents O or NH; at least one of X and Y represents an electron -withdrawing group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a substituted amide group and a cyano group; and the remainder of X and Y represents a hydrogen atom;

wherein R1 is a hydrogen atom; at least one of R2 and R3 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R2 and R3 represents a hydrogen atom or a cyano group;

wherein R4 is a hydrogen atom or a lower alkyl group; at least one of R5 and R6 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R5 and R6 represents a hydrogen atom or a cyano group;

wherein R7 is a hydrogen atom or a lower alkyl group; at least one of R8 and R9 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R8 and R9 represents a hydrogen atom or a cyano group;

wherein R10 is a hydrogen atom or a lower alkyl group; at least one of R11 and R12 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R11 and R12 represents a hydrogen atom or a cyano group; at the 5'-end or 3'-end as a base moiety, and
a nucleic acid to be photoligated having a base having a carbon-carbon double bond at the 3'-end or 5'-end as a base moiety capable of being photoligated to the photoligating nucleic acid,
wherein any one of the photoligating nucleic acid and the nucleic acid to be photoligated has a labeling moiety, while the other one not having a labeling moiety between the photoligating nucleic acid and the nucleic acid to be photoligated is immobilized on a substrate in advance;
a nucleic acid having a base sequence that is complementary to the base sequence of the target nucleic acid having a specific base sequence, can be generated by photoligation of the photoligating nucleic acid and the nucleic acid to be photoligated; and
the nucleic acid that can be generated by photoligation of the photoligating nucleic acid and the nucleic acid to be photoligated, can be washed under a washing condition capable of dissociating complementary double strands,
wherein the method comprises the following steps of:
hybridizing the target nucleic acid having a specific base sequence contained in a nucleic acid mixture with the photoligating nucleic acid and the nucleic acid to be photoligated;
immobilizing a nucleic acid having a labeling moiety on a substrate by photoirradiating the nucleic acid to result in photoligation of the nucleic acid with the photoligating nucleic acid and nucleic acid to be photoliqated that have been brought adjacent in a manner capable of photoligating through the hybridization using the target nucleic acid having a specific base sequence as the template;
removing any nucleic acid having a labeling moiety and not immobilized on the substrate through photoligation by washing the nucleic acid having a labeling moiety and immobilized on the substrate through photoligation, under a washing condition capable of dissociating hybridized complementary double strands; and
detecting the labeling moiety so as to detect the nucleic acid having a labeling moiety and immobilized on the substrate through photoligation;
wherein the washing condition involves washing at a washing temperature in the range of 80 deg. C. to 100 deg. C.;
wherein the process of hybridization and the process of immobilizing a nucleic acid having a labeling moiety through photoligation on the substrate are carried out in a reaction solution containing a salt having buffering effects;
wherein the reaction solution contains 20% to 60% v/v of a water-dispersible organic solvent; and
wherein the salt having buffering effects is a cacodylic acid salt.
[claim2]
2. The method according to claim 1 wherein the base moiety is a group represented by Formula I, and in the Formula I, Z is O; X is a hydrogen atom; and Y is a carboxyl group, a lower alkoxycarbonyl group, a substituted amide group or a cyano group.
[claim3]
3. The method according to claim 1 wherein the base moiety is a group represented by Formula II, and in the Formula II, at least one of R2 and R3 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R2 and R3 is a hydrogen atom.
[claim4]
4. The method according to claim 1 wherein the base moiety is a group represented by Formula III, and in the Formula III, at least one of R5 and R6 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R5 and R6 is a hydrogen atom.
[claim5]
5. The method according to claim 1 wherein the base moiety is a group represented by Formula IV, and in the Formula IV, at least one of R8 and R9 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R8 and R9 is a hydrogen atom.
[claim6]
6. The method according to claim 1 wherein the base moiety is a group represented by Formula V, and in the Formula V, at least one of R11 and R12 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R11 and R12 is a hydrogen atom.
[claim7]
7. The method according to claim 1 which comprises any label selected from the group consisting of biotin labels, dye labels and RI labels as a labeling moiety.
[claim8]
8. The method according to claim 1 wherein either one of photoligating nucleic acid and the nucleic acid to be photoligated which is immobilized on a substrate in advance Is immobilized on the substrate via a linker moiety.
[claim9]
9. The method according to claim 8 wherein the linker moiety comprises polyethylene glycol or an alkane.
[claim10]
10. The method according to claim 1 wherein the substrate is any one selected from the group consisting of a glass plate, CPG, and polystyrene beads.
[claim11]
11. The method according to claim 1 wherein the base ,having a carbon-carbon double bond is cytosine, thymine or uracil.
[claim12]
12. The method according to claim 1 wherein the photoligating nucleic acid is an oligonucleotide.
[claim13]
13. The method according to claim 1 wherein the photoligating nucleic acid is a DNA.
[claim14]
14. The method according to claim 1 wherein the photoligating nucleic acid is an RNA.
[claim15]
15. The method according to claim 1 wherein the photoligating nucleic acid is a peptide nucleic acid.
[claim16]
16. The method according to claim 1 wherein the nucleic acid to be photoliaated is an oligonucleotide.
[claim17]
17. The method according to claim 1 wherein the nucleic acid to be photoliaated is a DNA.
[claim18]
18. The method according to claim 1 wherein the nucleic acid to be photoligated is an RNA.
[claim19]
19. The method according to claim 1 wherein the nucleic acid to be photoliaated is a peptide nucleic acid.
[claim20]
20. The method according to claim 1 wherein the photoligating nucleic acid and the nucleic acid to be photoliaated are nucleic acids of the same kind.
[claim21]
21. The method according to claim 1 wherein a DNA microarray having either one of the photoligating nucleic acid and the nucleic acid to be photoliaated, which does not have a labeling moiety and is immobilized on a substrate, is used together with the other one of the photoligating nucleic acid and the nucleic acid to be photoligated, which is not immobilized on a substrate and has a labeling moiety.
[claim22]
22. The method according to claim 1 wherein the process of hybridization and the process of immobilizing a nucleic acid having a labeling moiety on a substrate by photoligation are carried out at a temperature of 20 deg. C. to 30 deg. C.
[claim23]
23. The method according to claim 1 wherein the pH of the reaction solution is in the range of 6.5 to 8.5.
[claim24]
24. The method according to claim 1 wherein the concentration of the salt having buffering effects is in the range of 5 to 250 mM.
[claim25]
25. The method according to claim 1 wherein the water-dispersible organic solvent is acetonitrile.
[claim26]
26. The method according to claim 1 wherein the reaction solution contains a salt of an alkali metal and/or alkaline earth metal, and the concentration of the salt is optimized with respect to the concentration of the target nucleic acid.
[claim27]
27. The method according to claim 26 which comprises sodium chloride and/or magnesium chloride as the salt of alkali metal and/or alkaline earth metal.
[claim28]
28. The method according to claim 1 wherein the photoirradiating is performed by irradiating with light having a wavelength of 330 nm or longer.
[claim29]
29. The method according to claim 1 wherein the washing condition involves washing at a washing temperature in the range of 95 deg. C. to 100 deg. C.
[claim30]
30. The method according to claim 1 wherein the washing condition involves washing with a washing solution containing a denaturing agent.
[claim31]
31. The method according to claim 1 wherein the washing condition involves washing with a washing solution containing a surfactant.
[claim32]
32. The method according to claim 1 wherein the label of the labeling moiety is a fluorescent dye label, and the process of detecting the labeling moiety includes process of performing fluorescence measurement using a laser scanner.
[claim33]
33. The method according to claim 1 wherein the label of the labeling moiety is a biotin label, and the process of detecting the labeling moiety includes process of performing a biotin-avidin binding reaction using a fluorescent dye-labeled avidin and process of performing fluorescence measurement using a laser scanner.
[claim34]
34. The method according to claim 1 for detecting point mutation in the base sequence of the target nucleic acid by using a photoligating nucleic acid and a nucleic acid to be photoligated, wherein a nucleic acid having a sequence having a single base substitution from a base sequence that is complementary to the base sequence of the target nucleic acid having a specific base sequence comprises a base sequence that would be generated by photoligation of the photoligating nucleic acid and the nucleic acid to be photoligated.
[claim35]
35. A method for detecting a target nucleic acid having a specific base sequence contained in a nucleic acid mixture, comprising: utilizing a set of nucleic acids for detecting a target nucleic acid having a specific base sequence, which the set of nucleic acids includes a photoligating nucleic acid comprising a nucleic acid having a group represented by the following Formula I, Formula II, Formula III, Formula IV or Formula V:

wherein Z represents O or NH; at least one of X and Y represents an electron -withdrawing group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a substituted amide group and a cyano group; and the remainder of X and Y represents a hydrogen atom;

wherein R1 is a hydrogen atom; at least one of R2 and R3 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R2 and R3 represents a hydrogen atom or a cyano group;

wherein R4 is a hydrogen atom or a lower alkyl group; at least one of R5 and R6 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group,. an amide group, a cyano group and a hydrogen atom; and the remainder of R5 and R6 represents a hydrogen atom or a cyano group;

wherein R7 is a hydrogen atom or a lower alkyl group; at least one of R8 and R9 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R8 and R9 represents a hydrogen atom or a cyano group;

wherein R10 is a hydrogen atom or a lower alkyl group; at least one of R11 and R12 represents a group selected from the group consisting of a carboxyl group, a lower alkoxycarbonyl group, a lower alkenyl group, a lower alkynyl group, a substituted amide group, an amide group, a cyano group and a hydrogen atom; and the remainder of R11 and R12 represents a hydrogen atom or a cyano group; at the 5'-end or 3'-end as a base moiety, and
a nucleic acid to be photoligated having a base having a carbon -carbon double bond at the 3'-end or 5'-end as a base moiety capable of being photoligated to the photoligating nucleic acid,
wherein any one of the photoligating nucleic acid and the nucleic acid to be photoligated has a labeling moiety, while the other one not having a labeling moiety between the photoligating nucleic acid and the nucleic acid to be photoligated is immobilized on a substrate in advance;
a nucleic acid having a base sequence that is complementary to the base sequence of the target nucleic acid having a specific base sequence, can be generated by photoligation of the photoligating nucleic acid and the nucleic acid to be photoligated; and
the nucleic acid that can be generated by photoligation of the photoligating nucleic acid and the nucleic acid to be photoligated, can be washed under a washing condition capable of dissociating complementary double strands,
wherein the method comprises steps of:
hybridizing the target nucleic acid having a specific base sequence contained in a nucleic acid mixture with the photoligating nucleic acid and the nucleic acid to be photoligated;
immobilizing a nucleic acid having a labeling moiety on a substrate by photoirradiating the nucleic acid to result in photoligation of the nucleic acid with the photoligating nucleic acid and nucleic acid to be photoligated, that have been brought adjacent in a manner capable of photoligating through the hybridization using the target nucleic acid having a specific base sequence as the template;
removing any nucleic acid having a labeling moiety and not immobilized on the substrate through photoligation by washing the nucleic acid having a labeling moiety and immobilized on the substrate through photoligation, under a washing condition capable of dissociating hybridized complementary double strands; and
detecting the labeling moiety so as to detect the nucleic acid having a labeling moiety and immobilized on the substrate through photoligation.
[claim36]
36. The method of claim 35 wherein the process of hybridization and the process of immobilizing a nucleic acid having a labeling moiety through photoligation on the substrate are carried out in a reaction solution containing a salt having buffering effects; and the salt having buffering effects is a cacodylic acid salt.
[claim37]
37. The method of claim 1, wherein the photoligating nucleic acid comprises a nucleic acid that is a nucleic acid or a peptide nucleic acid.
[claim38]
38. The method of claim 7 wherein the dye labels include fluorescent dye labels.
[claim39]
39. The method of claim 21, wherein said one of the photoligating nucleic acid and the nucleic acid to be photoligated, which is immobilized on a substrate, is a nucleic acid-or a peptide nucleic acid.
[claim40]
40. The method of claim 35, wherein the photoligating nucleic acid comprises a nucleic acid that is a nucleic acid or a peptide nucleic acid.
  • Inventor, and Inventor/Applicant
  • FUJIMOTO KENZO
  • YOSHIMURA YOSHINAGA
  • OGASAWARA SHINZI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
Reference ( R and D project ) CREST Nano Virtual Lab (Virtual Laboratory in Nanotechnology Areas) Creation of Innovative Technology by Integration of Nanotechnology with Information. Biological, and Environmental Technologies AREA
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