METHOD OF DETECTING NUCLEOTIDE SEQUENCE WITH AN INTRAMOLECULAR PROBE

• US 20090004666A1
• Filed: 07/14/2008
• Published: 01/01/2009
• Est. Priority Date: 01/20/2006
• Status: Active Grant

• Current Assignee: Olympus Corporation
• Original Assignee: Olympus Corporation
• Inventors: MORIMOTO, Nobuhiko, TANABE, Tetsuya
• Granted Patent: US 8,236,498 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

A nucleotide sequence-detecting method, including preparing a first intramolecular detecting sequence having a sequence complementary to a first sequence located at a 3′-side of the detecting site contained in the nucleotide sample and a second intramolecular detecting sequence having a sequence complementary to a second sequence located at a 5′-side of the detecting site, preparing a detecting chain containing a sequence of the detecting chain by connecting the first intramolecular detecting sequence to the 3′ terminal of the nucleotide sample and the second intramolecular detecting sequence to the 5′ terminal, allowing intramolecular hybridization at two positions of the detecting chain, connecting the 3′ terminal of the first intramolecular detecting sequence to the 5′ terminal of the second intramolecular detecting sequence, obtaining a cyclic structure, detecting the desired sequence in the nucleotide sample from the cyclic structure.

Claims

1. A nucleotide sequence-detecting method, comprising:

(a) preparing a nucleotide sample;

(b) preparing a first intramolecular detecting sequence having a sequence complementary to a first sequence located at a 3′

-side of the detecting site contained in the nucleotide sample and a second intramolecular detecting sequence having a sequence complementary to a second sequence located at a 5′

-side of the detecting site, wherein at least one of the 3′

-terminal nucleotide of the first intramolecular detecting sequence and the 5′

-terminal nucleotide of the second intramolecular detecting sequence is modified in such a manner that they can bind to each other;

(c) preparing a detecting chain containing a sequence of the detecting site by connecting the first intramolecular detecting sequence to the 3′

terminal of the nucleotide sample and the second intramolecular detecting sequence to the 5′

terminal;

(d) allowing intramolecular hybridization at two positions of the detecting chain between the first sequence and the first intramolecular detecting sequence and between the second sequence and the second intramolecular detecting sequence;

(e) connecting the 3′

terminal of the first intramolecular detecting sequence to the 5′

terminal of the second intramolecular detecting sequence directly or indirectly;

(f) obtaining a cyclic structure by the connection (e); and

(g) detecting the desired sequence in the nucleotide sample from the cyclic structure.

2. The nucleotide sequence-detecting method according to claim 1, wherein a dumbbell structure is formed by means of the intramolecular hybridization in (d) and the connection in (e) between the 5′

and 3′

terminals of the dumbbell structure occurs by means selected from the group consisting of ligation and gap ligation.

3. The nucleotide sequence-detecting method according to claim 2, wherein formation of the dumbbell structure and the connection thereof in (e) are performed together with thermal cycling under a thermal cycling condition previously determined.

4. The nucleotide sequence-detecting method according to claim 1, wherein the connection in (c) is performed by means selected from the group consisting of PCR by using at least a pair of primers and gap ligation, and restriction-enzyme cleavage and subsequent adapter ligation by using at least a pair of adapters.

5. The nucleotide sequence-detecting method according to claim 1, further comprising digesting un-ring-closed nucleotides with an enzyme after obtaining the cyclic structure in (f).

6. The nucleotide sequence-detecting method according to claim 1, wherein the desired sequence is detected in (g) by detection of the cyclic structure by means selected from the group consisting of DNA microarray, fluorescent beads, electrophoresis and mass spectrometry.

7. The nucleotide sequence-detecting method according to claim 1, wherein the desired sequence is detected in (g) by detection of the cyclic structure by detecting the amplification product obtained by using an elongation reaction using a primer that can detect formation of the connecting region of the cyclic structure.

8. The nucleotide sequence-detecting method according to claim 1, wherein the 5′

-terminal of the second intramolecular detecting sequence is phosphorylated.

9. The nucleotide sequence-detecting method according to claim 1, wherein the 3′

terminal of the second intramolecular detecting sequence includes a primer sequence and the 5′

terminal of the first intramolecular detecting sequence is phosphorylated.

10. The nucleotide sequence-detecting method according to claim 1, wherein the connection in (f) is performed by means selected from the group consisting of chemical binding and biochemical binding.

11. The nucleotide sequence-detecting method according to claim 10, wherein the chemical binding is performed by photo-assisted reaction.

12. The nucleotide sequence-detecting method according to claim 10, wherein the biochemical binding is performed by enzyme-assisted reaction.

13. The nucleotide sequence-detecting method according to claim 1, wherein

the first intramolecular detecting sequence contains a sequence complementary to the first sequence located at the 3′

side and a tag sequence previously designed and allocated to carry information about the detection target at the detecting site, and/or

the second intramolecular detecting sequence contains a sequence complementary to the second sequence located at the 5′

side of the detecting site and additionally a tag sequence previously designed and allocated to carry information about the detection target at the detecting site.

14. The nucleotide sequence-detecting method according to claim 1, wherein the sequence is detected by means selected from the group consisting of SNP detection, gene expression measurement, methylation detection, and detection of deletion, insertion, substitution and microsatellite.

16. A detection kit for use in the method according to claim 1, comprising any or all of enzymes, nucleotides, substrates, a buffer, and a detection microarray.

15. An intramolecular detecting sequence, comprising a first intramolecular detecting sequence containing a sequence complementary to a first sequence located at a 3′

side of a detecting site contained in a nucleotide sample, and a second intramolecular detecting sequence containing a sequence complementary to a second sequence located at a 5′

side of the detecting site, wherein at least one of the 3′

-sided nucleotide of the first intramolecular detecting sequence and the 5′

-terminal nucleotide of the second intramolecular detecting sequence is modified to become mutually bindable.

17. A nucleotide mutation-analyzing method, comprising:

(a) preparing a duplicated chain complementary to an analyte nucleotide and connecting sequences complementary to the mutation to be detected on the duplicated chain or to the region around the mutation to both terminals of the duplicated chain, wherein

these complementary sequences are different from each other and are so located to hybridize to the position on the duplicated chain between its terminal and the mutation or between the terminal and the mutation including mutation;

(b) making the single-stranded duplicated chain have an intramolecular structure containing at least two bending regions;

(c) making the terminals of the duplicated chain in the structure form a closed-ring nucleotide molecule covalently bound, directly or via a nucleoside monomer or a nucleotide different from mutation, by an oxygen or chemical reaction when there is an analyte mutation present;

(d) preparing a sequence containing the connecting region of the closed-ring nucleotide molecule or its complementary chain sequence, or both of them; and

(e) analyzing nucleotide mutation by detecting presence of the sequence containing the region where the prepared closed-ring nucleotide molecule is connected or its complementary chain sequence.

18. The nucleotide mutation-analyzing method according to claim 17, comprising:

(a) amplifying an analyte nucleotide first under a condition allowing amplification (first amplification), wherein

the primers used consist of first and second primers for amplification of the sequence containing the mutation sequence of the analyte nucleotide,

the first primer contains a second single-stranded sequence of the 3′

side of the mutation possibly containing the mutation to be detected in the analyte nucleotide (second-primer elongation chain), a first probe sequence homologous to the 3′

-sided sequence containing the mutation at the 5′

-terminal side of the first primer, and additionally, a first priming sequence complementary to the partial sequence at the 3′

side of the sequence corresponding to the first probe sequence on the second single-strand chain to the mutation site in the second single-strand chain, on the 3′

terminal of the first primer,

the second primer contains an elongation chain of the first primer, i.e., a second probe sequence homologous to the first single-stranded sequence of the 3′

side of the mutation, at the 5′

-terminal side of the second primer and a second priming sequence complementary to a partial sequence to the 3′

side of a sequence corresponding to the second probe sequence at the 3′

terminal of the second primer on the first single-strand chain to the first single-stranded the mutation site, and

the 5′

terminal of the second primer is phosphorylated;

(b) converting the first amplification product obtained by the first amplification into a single-stranded chain;

(c) making the single-strand-chain first amplification product form an intramolecular structure in ring-closure reaction, to give a closed-ring nucleotide molecule;

(d) amplifying the closed-ring nucleotide molecule obtained secondly under a condition allowing amplification (second amplification), to give a second amplification product containing the mutation to be detected contained in the closed-ring nucleotide molecule and sequences derived from at least the first and second probe sequences contained in the same closed-ring nucleotide molecule; and

(e) analyzing the mutation to be detected in the analyte nucleotide by detecting the second amplification product obtained.

19. The nucleotide mutation-analyzing method according to claim 18, wherein the base bound to the 3′

terminal of the first amplification product is eliminated and the terminal is smoothed after the first amplification (a).

20. The method according to claim 17, further comprising decomposing straight chain nucleotide molecules other than the closed-ring nucleotide molecule partially or completely.

21. The method according to claim 17, wherein the first and second primers have an artificially designed sequence usable for identification and/or amplification between the priming sequence and the probe sequence.

22. The method according to claim 17, wherein:

the first and second primers have an artificially designed sequence between the priming sequence and the probe sequence;

the artificially designed sequence is one or more sequences corresponding to or common to the mutation to be detected;

there is a second primer for each mutation site;

it is the first primer of the mutation at the mutation site;

these primers have different artificially designed sequences; and

multiple mutations are detected and analyzed simultaneously.

23. The method according to claim 17, wherein:

the first and second primers have an artificially designed sequence between the priming sequence and the probe sequence;

the first amplification is performed by using these primers;

the first amplification product obtained is amplified further (second amplification);

the probe sequence and the artificially designed sequence are detected; and

nucleotide mutation is analyzed based on the information thus obtained.

24. The method according to claim 17, wherein:

in the single-stranded nucleotide containing the mutation to be detected contained in the first amplification product, the 3′ and

5′

terminals of the single-stranded nucleotide hybridize intramolecularly to the mutation site or the region around it to form an intramolecular structure, and the gap or nick of the terminal ring is present in the region close to the mutation site in the intramolecular structure or the region close to the mutation site including the mutation site.

25. The method according to claim 24, wherein there is a nick between the mutation site of the intramolecular structure and the neighboring base and a closed-ring nucleotide molecule is formed there by action of a ligase.

26. The method according to claim 24, wherein there is a gap in the intramolecular structure, and a closed-ring nucleotide molecule is formed there by complementary chain synthesis by a polymerase and ligation by a ligase.

27. The method according to claim 17, wherein

the gap is a gap sequence having a sequence complementary to the mutation site of the first chain and the sequence close to the mutation site or a sequence complementary to the mutation site,

the first primer contains a first probe sequence having a sequence complementary to part of the continuous sequence present at the 5′

side of the first single-stranded mutation site on the 5′

-terminal side of the first primer,

the second primer contains a second probe sequence complementary to part of the continuous sequence close to the 5′

side of the sequence corresponding to the mutation site on the second single-strand chain on the 5′

side of the second primer, and

a closed-ring nucleotide molecule is formed by action of a ligase in the presence of a 5′

-terminal-phosphorylated fragment nucleotide having the nucleotide sequence of the gap sequence, after the first amplification by using the first and second primers.

28. The method according to claim 27, wherein the mutation to be detected is a single-nucleotide mutation.

29. The method according to claim 17, wherein:

the mutation to be detected is a single-nucleotide mutation;

the first primer contains a first probe sequence homologous to the first single-stranded mutation base and part of the continuous sequence present at the 3′

side of the mutation base on the 5′

-terminal side of the first primer; and

the second primer contains a second probe sequence homologous to part of the sequence close to the 3′

side of the second single-strand-chain mutation base on the 5′

side of the second primer.

30. The method according to claim 17, wherein

the first primer used in the second amplification has an identifiable first chemical labeling substance and the second primer has an identifiable second chemical labeling substance,

the second amplification product obtained is allowed to react with first particles carrying an antibody to the first chemical labeling substance and second particles carrying an antibody to the second chemical labeling substance, and

the mutation to be detected in the analyte nucleotide is analyzed by detecting the particle aggregation caused by the reaction.

31. The method according to claim 17, wherein

first and/or second primers previously labeled with a fluorescent dye are used in the second amplification,

the second amplification product obtained is allowed to hybridize to a nucleotide microarray carrying a probe for capturing the second amplification product, and

the mutation to be detected in the analyte nucleotide is analyzed by detecting the fluorescent dye label-derived fluorescent dye on the nucleotide microarray.

32. The method according to claim 22, wherein

the artificially designed sequences are pre-selected so that the multiple identifiable fluorescent dyes are correlated with the mutation types possibly observed at the same mutation site,

the sequence of the artificially designed sequences are designed so that the mutated nucleotides containing multiple mutation types possibly observed at the same mutation site hybridize to the same probes immobilized on a microarray previously made available, and

the mutation to be detected in the analyte nucleotide is analyzed by detecting the artificially designed sequences selected and designed as described above and labeled with fluorescent dyes in multiple colors on a nucleotide microarray.

33. The method according to claim 22, wherein

first and/or second primers previously labeled with a fluorescent dye are used in the second amplification,

the second amplification product obtained is allowed to hybridize to fluorescent-identifiable particles, each carrying a probe for capturing one kind of artificially designed sequence, and

the mutation to be detected in the analyte nucleotide is analyzed, based on the information on the fluorescence from the fluorescence particle and the second amplification product.

34. The method according to claim 33, wherein the fluorescence particle is selected from the group consisting of bead and quantum dot containing a fluorescent dye and bead containing multiple kinds of quantum dots.

37. The method according to claim 17, wherein the amplification method is a PCR method.

38. The method according to claim 37, wherein the second amplification method is asymmetric PCR.

39. An assay kit for performing the method according to claim 17, comprising a primer set, enzymes, substrates, a buffer agent, labeling substances, probes and/or a nucleotide microarray.

40. The assay kit according to claim 39, further comprising fluorescence particles.

35. A nucleotide mutation-analyzing method, comprising:

(a) amplifying an analyte nucleotide first under a condition allowing amplification (first amplification), wherein

primers used include first and second primers for amplification of a sequence containing the mutation sequence of the analyte nucleotide,

the first primer contains a second single-stranded sequence of a 3′

side of the mutation possibly containing the mutation to be detected in the analyte nucleotide (second-primer elongation chain) or a first probe sequence homologous to the 3′

-sided sequence containing the mutation at a 5′

-terminal side of the first primer, and additionally, a first priming sequence complementary to the partial sequence at the 3′

side of the sequence corresponding to the first probe sequence on the second single-strand chain to the mutation site in the second single-strand chain, on the 3′

terminal of the first primer, and

the second primer contains a second probe sequence homologous to the first single-stranded sequence of the 3′

side of the mutation, an elongation chain of the first primer, at the 5′

-terminal side of the second primer, and a second priming sequence complementary to a partial sequence at the 3′

side of a sequence corresponding to the second probe sequence at the 3′

terminal of the second primer on the first single-strand chain to the first single-stranded mutation site, and the 5′

terminal of the second primer is phosphorylated;

(b) converting the first amplification product obtained by the first amplification into a single-stranded chain;

(c) making the single-strand-chain first amplification product form an intramolecular structure in ring-closure reaction, to give a closed-ring nucleotide molecule; and

(d) the mutation to be detected in the analyte nucleotide is analyzed by detecting the difference in conformation between the closed-ring nucleotide molecule and non-ring-closed straight chain nucleotide molecules.

36. The method according to claim 35, wherein the difference in conformation between the closed-ring nucleotide molecule and the non-ring-closed straight chain nucleotide molecules is detected by an electrophoretic method.

41. A nucleotide sequence-detecting method, comprising:

(a) preparing a nucleotide sample;

(b) preparing a first detecting chain-preparing nucleotide containing a sequence complementary to a first nucleotide sequence located at a 3′

side of the detecting site contained in the nucleotide sample and a primer sequence on a 5′

side that hybridizes to the more 3′

-side than the first nucleotide sequence, and a second detecting chain-preparing nucleotide containing a sequence complementary to a second sequence located at the 5′

side of the detecting site, a complementary chain synthesis-inhibiting structure on the 3′

side thereof, and an oligonucleotide sequence on the 3′

side that hybridizes to the more 5′

-side than the second sequence,

wherein at least one of the 5′

-terminal nucleotide of the first detecting chain-preparing nucleotide and the 3′

-terminal nucleotide of the second detecting chain-preparing nucleotide is modified in such a manner that they can bind to each other;

(c) preparing a detecting chain by allowing the primer sequence of the first detecting chain-preparing nucleotide and the oligonucleotide sequence of the second detecting chain-preparing nucleotide to hybridize to the nucleotide sample, allowing elongation reaction of the second detecting chain-preparing nucleotide with its complementary chain, and allowing ligation reaction at the 5′

terminal of the first detecting chain-preparing nucleotide;

(d) allowing intramolecular hybridization of the detecting chain at two positions between the first sequence and the first detecting chain-preparing nucleotide and between the second sequence and the second detecting chain-preparing nucleotide;

(e) forming a cyclic structure by ring closure of the detecting chain at the 3′

terminal of the first detecting chain-preparing nucleotide and the 5′

terminal of the second detecting chain-preparing nucleotide;

(f) amplifying the sequence containing the connecting region of the cyclic structure; and

(g) detecting the detecting-site sequence in the nucleotide sample by detecting the amplification product obtained by amplification (f).

42. The nucleotide sequence-detecting method according to claim 41, wherein the ligation and ring closure in (e) is performed by means selected from the group consisting of ligation and gap ligation.

43. The method according to claim 42, wherein the steps of intramolecular hybridization and ligation/ring closure are performed together with thermal cycling under a thermal cycling condition previously determined.

44. The method according to claim 41, further comprising digesting non-ligated/ring-closed nucleotides after the ligation and ring closure.

45. The method according to claim 41, wherein the amplification step (f) is PCR producing a product containing the connecting region.

46. The method according to claim 41, wherein the amplification step (f) is RNA synthesis by in-vitro transcription with an RNA polymerase, producing a product containing the connecting region.

47. The method according to claim 41, wherein the modification allowing ligation is phosphorylation of nucleotide.

48. The method according to claim 41, wherein the step (g) is performed by detecting hybridization between the amplification product and a DNA microarray

49. The method according to claim 41, wherein the step (g) is performed by indirect detection of the amplification product, i.e., by hybridization to the amplification product, hybridization of the amplification product to a nucleotide carrying a detectable label, and detection of the detectable label.

50. A detection kit for use in the method according to claim 41, comprising a reagent containing any or all of enzymes, nucleotides, substrates, a buffer, and a detection microarray.