Probe synthesis method for nucleic acid detection

Abstract

The present invention relates to a probe synthesis method and a probe synthesis kit for nucleic acid detection, which are intended for the fluorophore labeling and detection of a target gene. In this method, a probe unit for analyte nucleic acid recognition having a base sequence specific to an analyte nucleic acid and plural labeled probe units each having a fluorophore labeled internal base are hybridized to oligonucleotides complementary thereto, and the adjacent probe units are bonded by ligation. As a result, an analytical probe with increased fluorescence intensity is synthesized easily and inexpensively, while the number of fluorophore labels is controlled.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing procedures to synthesize a probe labeled with plural fluorophores by ligation and detect a target molecule by the hybridization of the probe thereto;

FIG. 2 is a diagram showing procedures to synthesize a probe labeled with plural fluorophores by the hybridization of oligonucleotides (probe units) to complementary oligonucleotides and subsequent ligation;

FIG. 3 is a diagram showing procedures to synthesize a probe labeled with plural fluorophores by the linking of fluorophore unlabeled oligonucleotides (probe units to be labeled) by ligation and subsequent fluorophore labeling;

FIG. 4 is a diagram showing procedures to synthesize a probe labeled with plural fluorophores by the hybridization of fluorophore labeled oligonucleotides (labeled probe units) to complementary oligonucleotides, gap filling by use of DNA polymerase, and subsequent ligation;

FIG. 5 is a diagram showing procedures to synthesize a probe comprising plural fluorophores of plural kinds introduced therein by the linking of oligonucleotides (labeled probe units) labeled with various fluorophores;

FIG. 6 is a diagram showing procedures to synthesize a branched probe labeled with plural fluorophores by the ligation of oligonucleotides (labeled probe units) to the branch portions of the branched probe;

FIG. 7 is a diagram showing procedures to synthesize a probe labeled with plural fluorophores by the hybridization of fluorophore labeled oligonucleotides (labeled probe units) to complementary oligonucleotides and subsequent ligation;

FIG. 8 is a graph showing the comparison in fluorophore intensity between a probe labeled with a single fluorophore and a probe labeled with plural fluorophores;

FIG. 9 is a graph showing the relationship between the position of a fluorophore labeled base and ligation efficiency in the ligation of oligonucleotides (labeled probe units) each having a fluorophore labeled internal base;

FIG. 10 is a diagram showing procedures to synthesize a probe labeled with plural fluorophores by the hybridization of biotin labeled oligonucleotides (labeled probe units) to complementary oligonucleotides, gap filling by use of DNA polymerase, and subsequent ligation, followed by fluorophore labeling; and

FIG. 11 is a fluorescence microscope image showing the measurement of a probe labeled with one quantum dot and a probe labeled with plural quantum dots.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention will be described in detail with reference to drawings.

FIG. 1 is a diagram showing procedures to synthesize a fluorophore labeled probe by the present invention and detect a target molecule by the hybridization of the probe thereto. Reference numeral 1 denotes an analyte (or target) molecule (analyte nucleic acid), and reference numeral 2 denotes an oligonucleotide (probe unit for analyte nucleic acid recognition) designed to hybridize to the analyte molecule. Reference numerals 3, 4, 5, and 6 denote oligonucleotides (labeled probe units) each having a fluorophore labeled base that is located internal except at the 5′-end and the 3′-end. The oligonucleotides 2, 3, 4, 5, and 6 are linked by ligation to thereby obtain an oligonucleotide probe 7 labeled with plural fluorophores. The analyte molecule is detected by the hybridization of the oligonucleotide probe 7 thereto.

The length of an analyte molecule hybridizing portion in the sequence of the oligonucleotide is not particularly limited and is preferably approximately 18 to 25 nucleotides. This is because the existing probability of a sequence a little less than 20 nucleotides in length is one or less in a random base sequence having a length approximately equal to that of the human genome and as a result, the oligonucleotide can specifically recognize the analyte molecule. Moreover, the length of the fluorophore labeled oligonucleotide is not particularly limited and is preferably approximately 9 to 30 nucleotides.

FIG. 2 is a schematic diagram specifically showing ligation reaction. An oligonucleotide (probe unit for analyte nucleic acid recognition) hybridizing to an analyte molecule and fluorophore labeled oligonucleotides (labeled probe units) are hybridized to oligonucleotides (complementary oligonucleotides) each having a sequence complementary thereto to thereby form double-stranded DNA. Then, the linking between the oligonucleotide hybridizing to an analyte molecule and the fluorophore labeled oligonucleotide adjacent thereto and between the adjacent fluorophore labeled oligonucleotides is performed by ligation to thereby synthesize an oligonucleotide probe labeled with plural fluorophores. Reference numeral 2 denotes an oligonucleotide (probe unit for analyte nucleic acid recognition) designed to hybridize to the analyte molecule. Reference numerals 3, 4, 5, and 6 denote oligonucleotides (labeled probe units) each having a fluorophore labeled base that is located internal except at the 5′-end and the 3′-end. Reference numeral 11 denotes an oligonucleotide (complementary oligonucleotide) having a sequence complementary to a portion of the sequences of the oligonucleotides 2 and 3; reference numeral 12 denotes an oligonucleotide (complementary oligonucleotide) having a sequence complementary to a portion of the sequences of the oligonucleotides 3 and 4; reference numeral 13 denotes an oligonucleotide (complementary oligonucleotide) having a sequence complementary to a portion of the sequences of the oligonucleotides 4 and 5; and reference numeral 14 denotes an oligonucleotide (complementary oligonucleotide) having a sequence complementary to a portion of the sequences of the oligonucleotides 5 and 6. The linking between the oligonucleotides 2 and 3, between the oligonucleotides 3 and 4, between the oligonucleotides 4 and 5, and between the oligonucleotides 5 and 6 is performed by ligation to thereby synthesize an oligonucleotide probe 7 labeled with plural fluorophores.

The fluorophore labeled oligonucleotides 3, 4, 5, and 6 and the complementary oligonucleotide strands 11, 12, 13, and 14 are designed so that they are hybridized only to their intended sequences to prevent mishybridization or so that their Tm values (melting temperatures) serving as an index for hybridization stability are kept as equal as possible. The ligation may be performed sequentially in such a way that the oligonucleotides 2 and 3 are linked, then, the oligonucleotide 4 is linked to the linked oligonucleotides, and further, the oligonucleotide 5 is linked thereto, or may be performed at once. Moreover, only the fluorophore labeled oligonucleotide strands may be linked as shown in FIG. 2(A), while both the fluorophore labeled oligonucleotide strands and the complementary strands may be linked as shown in FIG. 2(B).

The positions of the fluorophore labels in the fluorophore labeled oligonucleotides 3, 4, 5, and 6 are set to a position five or more bases internal from the 5′-end and the 3′-end, because the positions of the fluorophore labels within the 2nd to 4th bases from both the ends reduce ligation efficiency. This may be because the steric hindrance of the fluorophores and linkers influences enzyme reaction. Since the same tendency is observed at both the 5′-end and the 3′-end, the positions of the fluorophore labels are set to bases located internal except at both the ends to at the 4th base position therefrom. Therefore, the fluorophore labels in the fluorophore labeled oligonucleotide probe obtained after the ligation are placed at a distance of at least eight or more bases.

The timing of fluorophore labeling for oligonucleotides may be either before or after ligation. Fluorophore labeled oligonucleotides may be linked to thereby synthesize an oligonucleotide probe labeled with plural fluorophores, as shown in FIGS. 1 and 2, while oligonucleotides (probe units to be labeled) 23, 24, 25, and 26 each having a linker 21 for fluorophore introduction may be linked to thereby synthesize an oligonucleotide 27, followed by introduction of fluorophores 28 into the linker portions to thereby synthesize a probe 29, as shown in FIG. 3. Examples of the linkers for fluorophore introduction include, but not limited to, biotin, an amino group, and a thiol group. The position of the linker is set to a base located internal except at both the ends to at the 4th base position therefrom, as with the fluorophore.

Alternatively, the fluorophore labeled oligonucleotides may be designed to produce 1-base or more gaps therebetween in their hybridization to the complementary oligonucleotides. In this case, unintended ligation products attributed to mishybridization can be prevented by gap filling by use of DNA polymerase and subsequent ligation. As shown in FIG. 4, an oligonucleotide (probe unit for analyte nucleic acid recognition) 2 hybridizing to an analyte molecule and fluorophore labeled oligonucleotides (labeled probe units) 33, 34, 35, and 36 are hybridized to oligonucleotides (complementary oligonucleotides) 11, 12, 13, and 14 complementary thereto. The sequences of the oligonucleotides 33, 34, 35, and 36 are designed to produce 1-base or more gaps therebetween in their hybridization to the oligonucleotides 11, 12, 13, and 14 complementary thereto. Next, the fluorophore labeled oligonucleotides 33, 34, 35, and 36 are elongated by use of DNA polymerase to thereby fill the gaps. As a result, they are converted to oligonucleotides 43, 44, 45, and 46. Then, ligation is performed to thereby obtain an oligonucleotide probe 7 labeled with plural fluorophores. In this case as well, the fluorophores may be introduced into linkers after the ligation:

As shown in FIGS. 5(A) and 5(B), oligonucleotides (labeled probe units) 56, 57, 58, and 59 respectively labeled with various fluorophores 51, 52, 53, and 54 and oligonucleotides (probe units for analyte nucleic acid recognition) 60 and 61 recognizing mutually different analyte molecules can be prepared and linked to thereby obtain probes 62 and 63 labeled with plural fluorophores of plural kinds. The fluorescence wavelength of the whole probe can be controlled by changing the kinds of fluorophores.

Since quantum dots can be excited in a wide wavelength range, the use of the quantum dots as fluorophores can easily synthesize probes having various fluorescence wavelengths. Plural analyte molecules can be distinguished on the basis of fluorescence wavelengths and tested by one measurement by changing the sequences recognizing the analyte molecules and changing the combination of fluorophores (quantum dots) used according to the sequences. When m kinds are selected and used from n kinds of fluorophores, _nC_m kinds of fluorescent probes can be synthesized.

For a branched probe as well, oligonucleotides (complementary oligonucleotides) complementary to probes in branch portions can be prepared, followed by ligation to thereby obtain an oligonucleotide comprising plural fluorophores introduced into each of the branch portions. As shown in FIG. 6, a branched oligonucleotide (probe unit for analyte nucleic acid recognition) 47 hybridizing to an analyte molecule can be linked to oligonucleotides (labeled probe units) 3, 4, 5, and 6 each having a fluorophore labeled base that is located internal except at the 5′-end and the 3′-end, to thereby introduce plural fluorophores into each of the branch portions. This can control the number of labels while synthesizing a branched probe 48 comprising plural fluorophores introduced therein.

As described above, an oligonucleotide (probe unit for analyte nucleic acid recognition) having a sequence recognizing an analyte molecule sequence and fluorophore labeled oligonucleotides (labeled probe units) can be linked by ligation to thereby control the number of fluorophores while synthesizing a probe labeled with plural fluorophores. To detect another analyte molecule, only the sequence recognizing an analyte molecule may be changed, and the fluorophore labeled portions can be utilized universally, irrespective of analyte molecule types. Specifically, the present invention also provides a kit for synthesis of these fluorophore labeled probes that can be utilized universally. The kit of the present invention may be intended for particular analyte molecule analysis or may be provided in a form wherein a user prepares an oligonucleotide having a sequence recognizing an analyte molecule and can easily synthesize fluorophore labeled probes for various analyte molecules. Fluorophore labeled oligonucleotides (labeled probe units), fluorophores, and linkers for fluorophore introduction into the oligonucleotides as essential components in the kit are as described above. The present invention synthesizes a probe comprising plural fluorophores introduced into a linear oligonucleotide or branched oligonucleotide strand. In this probe, the fluorophore labels of bases are placed at a distance of eight or more bases.

The kit of the present invention may comprise other reagents necessary for probe synthesis, in addition to the fluorophore labeled oligonucleotides, fluorophores, and linkers for fluorophore introduction into the oligonucleotides as essential components. Examples of such reagents can include reagents such as enzymes, buffers that provide conditions suitable to enzyme reaction, and substrates necessary for enzyme reaction. Furthermore, the kit may provide reagents necessary for one reaction dispensed into reaction containers.

EXAMPLES

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the present invention is not intended to be limited to these Examples.

Example 1

Synthesis Method of Oligonucleotide Probe Having Plural Fluorophore Labels by Ligation

This Example shows the synthesis of an oligonucleotide probe having plural fluorophore labels by use of a method of the present invention. Ten kinds of oligonucleotides (labeled probe units) 70, 71, 72, 73, 74, 75, 76, 77, 78, and 79 (SEQ ID NOs: 1 to 10) were prepared, each of which comprised an 18-base sequence and had internal T (thymidine) at the 10th base position labeled with a fluorophore FITC. Ten kinds of oligonucleotides (complementary oligonucleotides) 80, 81, 82, 83, 84, 85, 86, 87, 88, and 89 (SEQ ID NOs: 11 to 20) were prepared which hybridized to the 10 kinds of oligonucleotides (labeled probe units) to form a sticky end. Moreover, an oligonucleotide (probe unit for analyte nucleic acid recognition) 90 (SEQ ID NO: 21) was prepared which hybridized to both an analyte molecule and the oligonucleotide 80 prepared in advance. Each oligonucleotide sequence was designed to form double-stranded DNA, as shown in FIG. 7.

The oligonucleotides were mixed at each amount of 50 pmol and subjected to ligation through reaction at 37° C. for 60 minutes by use of Invitrogen T4 ligase to thereby obtain a probe 91 comprising the 10 kinds of fluorophore labeled oligonucleotides and the oligonucleotide for analyte molecule recognition linked together. The fluorophore labeled base in the fluorophore labeled oligonucleotide of 18 bases in length is located at the 10th base position. Therefore, the fluorophore labeled oligonucleotide has 9 fluorophore unlabeled bases at the 5′-end and 8 fluorophore unlabeled bases at the 3′-end. Thus, the probe obtained after the ligation has the fluorophore labeled bases every 18 bases.

After the completion of reaction, the ligase was inactivated, and the fluorescence intensity of the obtained solution was measured. FIG. 8 shows fluorescence intensity measurement results of the fluorophore labeled oligonucleotide 70 before reaction and the probe 91 obtained by reaction. Since plural fluorophores were introduced into one probe molecule, fluorescence intensity per unit concentration could be confirmed to be increased by 10 or more times.

FITC labeled oligonucleotide 70:
5′-aagtgacctttaaacatg-3′ (SEQ ID NO: 1)
FITC labeled oligonucleotide 71:
5′-aagtgagctttaaaccag-3′ (SEQ ID NO: 2)
FITC labeled oligonucleotide 72:
5′-aagtggtctttaaacgcg-3′ (SEQ ID NO: 3)
FITC labeled oligonucleotide 73:
5′-aagtgtactttaaactgg-3′ (SEQ ID NO: 4)
FITC labeled oligonucleotide 74:
5′-aagtgatctttaaacacg-3′ (SEQ ID NO: 5)
FITC labeled oligonucleotide 75:
5′-acttgacctttccacatg-3′ (SEQ ID NO: 6)
FITC labeled oligonucleotide 76:
5′-acttgagctttccaccag-3′ (SEQ ID NO: 7)
FITC labeled oligonucleotide 77:
5′-acttggtctttccacgcg-3′ (SEQ ID NO: 8)
FITC labeled oligonucleotide 78:
5′-acttgtactttccactgg-3′ (SEQ ID NO: 9)
FITC labeled oligonucleotide 79:
5′-acttgatctttccacacg-3′ (SEQ ID NO: 10)
oligonucleotide 80:
5′-ggtcacttgcacattt-3′   (SEQ ID NO: 11)
oligonucleotide 81:
5′-gctcacttcatgtttaa-3′  (SEQ ID NO: 12)
oligonucleotide 82:
5′-gaccacttctggtttaa-3′  (SEQ ID NO: 13)
oligonucleotide 83:
5′-gtacacttcgcgtttaa-3′  (SEQ ID NO: 14)
oligonucleotide 84:
5′-gatcacttccagtttaa-3′  (SEQ ID NO: 15)
oligonucleotide 85:
5′-ggtcaagtcgtgtttaa-3′  (SEQ ID NO: 16)
oligonucleotide 86:
5′-gctcaagtcatgtggaa-3′  (SEQ ID NO: 17)
oligonucleotide 87:
5′-gaccaagtctggtggaa-3′  (SEQ ID NO: 18)
oligonucleotide 88:
5′-gtacaagtcgcgtggaa-3′  (SEQ ID NO: 19)
oligonucleotide 89:
5′-gatcaagtccagtggaa-3′  (SEQ ID NO: 20)
oligonucleotide 90:
5′-                      (SEQ ID NO: 21)
ctagccgagtagtgttgggttttaaatgtgc-3′

Example 2

Difference in Ligation Efficiency Depending on Difference in Position of Fluorophore Label

Fluorophore labeled oligonucleotides were linked together by ligation in the same way as in Example 1 to thereby synthesize a probe labeled with plural fluorophores.

Of two fluorophore labeled oligonucleotides (labeled probe units) 95 and 96 to be linked, the oligonucleotide 96 was subjected to ligation using varying positions of its fluorophore label, as shown in FIG. 9(A). After the electrophoresis of the reaction products, the amounts of the ligation product 97 and the unreacted product 98 were measured with a densitometer to calculate ligation efficiency. As a result, when the fluorophore label of the oligonucleotide 96 was located at the 5′-end to at the fourth base position, ligation efficiency was low, demonstrating that these positions are not suitable for probe synthesis, as shown in FIG. 9(B). The same tendency was also observed at the 3′-end. This suggested that the position of a fluorophore label within the fourth bases from both the ends inhibits enzyme function and thereby reduces ligation efficiency. Therefore, the introduction position of a fluorophore label was set to a position five or more bases internal from the 5′-end and 3′-end of an oligonucleotide in subsequent Examples.

Example 3

Probe Synthesis Method Comprising Gap Filling Step Using DNA Polymerase Before Ligation

This Example shows probe synthesis by use of the method of the present invention, tcomprising the hybridization of oligonucleotides having plural fluorophore labels to complementary oligonucleotides, elongation reaction with DNA polymerase, and subsequent ligation.

Five kinds of oligonucleotides (labeled probe units) 100, 101, 102, 103, and 104 (SEQ ID NOs: 22 to 26) were prepared, each of which comprised a 16-base sequence and had internal T (thymidine) at the 9th base position labeled with biotin. Five kinds of oligonucleotides (complementary oligonucleotides) 110, 111, 112, 113, and 114 (SEQ ID NOs: 27 to 31) were prepared which hybridized to the five kinds of oligonucleotides (labeled probe units) prepared. Moreover, an oligonucleotide (probe unit for analyte nucleic acid recognition) 105 (SEQ ID NO: 32) was prepared which recognized and hybridized to an analyte molecule and the oligonucleotide 110. Each oligonucleotide sequence was designed to form double-stranded DNA but to produce an at least one-base or more gap, as shown in FIG. 10. The fluorophore labeled base in the fluorophore labeled oligonucleotide of 16 bases in length is located at the ninth base position. Therefore, the fluorophore labeled oligonucleotide has eight fluorophore unlabeled bases at the 5′-end and seven fluorophore unlabeled bases at the 3′-end. Thus, the probe obtained after the filling of two-base gaps and ligation has the fluorophore labeled bases every 18 bases.

The oligonucleotides were mixed at each amount of 50 pmol and elongated through reaction at 37° C. for 10 minutes in a buffer solution containing dNTP and DNA polymerase to thereby cause reaction for the filling of two-base gaps. After this elongation reaction, ligation was performed to thereby obtain a probe 106 comprising the biotin labeled oligonucleotides and the oligonucleotide for analyte molecule recognition linked together. In this procedure, complementary strands of the probe were also linked by the ligation to form a complementary strand 107. The probe 106 and quantum dots 108 labeled with streptavidin were mixed to thereby obtain a probe 109 labeled with plural quantum dots. FIG. 11 shows a result of measurement of the obtained probe with a fluorescence microscope. The probes that emitted fluorescence were observed in an image 110 showing the measurement of the probe 109, whereas no distinct fluorescent image was observed in an image 111 showing the measurement of a probe comprising one quantum dot introduced therein, demonstrating that the present invention can easily increase the florescence intensity of a probe.

biotin labeled oligonucleotide
100:
5′- gatgagctttggacca-3′          (SEQ ID NO: 22)
biotin labeled oligonucleotide
101:
5′- gatggtctttggacgc-3′          (SEQ ID NO: 23)
biotin labeled oligonucleotide
102:
5′- gatgtactttggactg-3′          (SEQ ID NO: 24)
biotin labeled oligonucleotide
103:
5′- gatgatctttggacac-3′          (SEQ ID NO: 25)
biotin labeled oligonucleotide
104:
5′- tctgacctttttacat-3′          (SEQ ID NO: 26)
oligonucleotide 110:
5′- gctcatctcatgtcca-3′          (SEQ ID NO: 27)
oligonucleotide 111:
5′- gaccatctctggtcca-3′          (SEQ ID NO: 28)
oligonucleotide 112:
5′- gtacatctcgcgtcca-3′          (SEQ ID NO: 29)
oligonucleotide 113:
5′- gatcatctccagtcca-3′          (SEQ ID NO: 30)
oligonucleotide 114:
5′- ggtcagatcgtgtcca-3′          (SEQ ID NO: 31)
oligonucleotide 105:
5′-
ctagccgagtagtgttgggttttatggacat-3′ (SEQ ID NO: 32)

The present invention can conveniently and inexpensively synthesize a probe labeled with plural fluorophores by linking oligonucleotides each having a fluorophore labeled internal base by ligation. This can achieve improvement in the performance of probes used for various fluorescence detections and in detection limit and allows for single-molecule measurement with sufficient sensitivity. Thus, the present invention is useful in wide fields from basic research to clinical fields, such as the quantification of trace amounts of nucleic acids and disease diagnosis using this quantification.

[Free Text of Sequence Listing]

• SEQ ID NO: 1—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 2—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 3—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 4—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 5—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 6—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 7—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 8—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 9—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 10—Description of artificial sequence: FITC labeled probe unit
• SEQ ID NO: 11—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 12—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 13—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 14—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 15—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 16—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 17—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 18—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 19—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 20—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 21—Description of artificial sequence: probe unit hybridizing to analyte molecule
• SEQ ID NO: 22—Description of artificial sequence: biotin labeled probe unit
• SEQ ID NO: 23—Description of artificial sequence: biotin labeled probe unit
• SEQ ID NO: 24—Description of artificial sequence: biotin labeled probe unit
• SEQ ID NO: 25—Description of artificial sequence: biotin labeled probe unit
• SEQ ID NO: 26—Description of artificial sequence: biotin labeled probe unit
• SEQ ID NO: 27—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 28—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 29—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 30—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 31—Description of artificial sequence: oligonucleotide complementary to probe unit
• SEQ ID NO: 32—Description of artificial sequence: probe unit hybridizing to analyte molecule

Claims

1. An analytical probe production method comprising: a first step wherein a probe unit for analyte nucleic acid recognition having a base sequence specific to an analyte nucleic acid and plural labeled probe units having mutually different base sequences and each being labeled with a fluorophore are hybridized to plural complementary oligonucleotides each having a sequence complementary to a portion of the neighborhood of the 5′-end of one of the probe units and to a portion of the neighborhood of the 3′-end of one of the other probe units to thereby obtain a hybridized probe unit comprising the probe unit for analyte nucleic acid recognition, the plural labeled probe units, and the plural complementary oligonucleotides; anda second step wherein the adjacent probe units in the hybridized probe unit are bonded by ligation to thereby obtain an analytical probe labeled with plural fluorophores.

2. The analytical probe production method according to claim 1, further comprising a step wherein DNA polymerase and substrate dNTP are allowed to act on the hybridized probe unit before the ligation to thereby fill the gaps between the probe units.

3. The analytical probe production method according to claim 1, wherein the fluorophore is bound with a base located five or more bases internal from the 5′-end and 3′-end of the labeled probe unit.

4. The analytical probe production method according to claim 1, wherein the fluorophore is bound with a base located substantially at the center of the labeled probe unit.

5. The analytical probe production method according to claim 1, wherein the labeled probe unit has a length between 9 nucleotides and 30 nucleotides inclusive.

6. The analytical probe production method according to claim 1, wherein the plural labeled probe units are labeled with the fluorophores that are mutually identical.

7. The analytical probe production method according to claim 1, wherein the plural labeled probe units are labeled with the fluorophores that are mutually different.

8. An analytical probe production method comprising: a first step wherein a probe unit for analyte nucleic acid recognition having a base sequence specific to an analyte nucleic acid and plural probe units to be labeled having mutually different base sequences are hybridized to plural complementary oligonucleotides each having a sequence complementary to a portion of the neighborhood of the 5′-end of one of the probe units and to a portion of the neighborhood of the 3′-end of one of the other probe units to thereby obtain a hybridized probe unit comprising the probe unit for analyte nucleic acid recognition, the plural probe units to be labeled, and the plural complementary oligonucleotides;a second step wherein the adjacent probe units in the hybridized probe unit are bonded by ligation; anda third step wherein a fluorophore is introduced into each of the plural probe units to be labeled to thereby obtain an analytical probe labeled with plural fluorophores.

9. The analytical probe production method according to claim 8, wherein the method further comprises a step wherein DNA polymerase and substrate dNTP are allowed to act on the hybridized probe unit before the ligation to thereby fill the gaps between the probe units.

10. The analytical probe production method according to claim 8, wherein the fluorophore is bound with a base located five or more bases internal from the 5′-end and 3′-end of the probe unit to be labeled.

11. The analytical probe production method according to claim 8, wherein the fluorophore is bound with a base located substantially at the center of the probe unit to be labeled.

12. The analytical probe production method according to claim 8, wherein the probe unit to be labeled has a length between 9 nucleotides and 30 nucleotides inclusive.

13. The analytical probe production method according to claim 8, wherein the fluorophores that are mutually identical are introduced into the plural probe units to be labeled.

14. The analytical probe production method according to claim 8, wherein the fluorophores that are mutually different are introduced into the plural probe units to be labeled.

15. The analytical probe production method according to claim 1, wherein the probe unit for analyte nucleic acid recognition is a branched probe having branches.

16. A kit for analytical probe production comprising: plural probe units having mutually different base sequences; and plural complementary oligonucleotides each having a sequence complementary to a portion of the neighborhood of the 5′-end of one of the probe units and to a portion of the neighborhood of the 3′-end of one of the other probe units.

17. The kit for analytical probe production according to claim 16, wherein one of the plural probe units is a probe unit for analyte nucleic acid recognition having a base sequence specific to an analyte nucleic acid.

18. The kit for analytical probe production according to claim 17, wherein the plural probe units other than the probe unit for analyte nucleic acid recognition are labeled probe units each labeled with a fluorophore.

19. The kit for analytical probe production according to claim 18, wherein the labeled probe unit has a length between 9 nucleotides and 30 nucleotides inclusive, and the fluorophore is bound with a base located five or more bases internal from the 5′-end and 3′-end of the labeled probe unit.

20. An analytical probe comprising: a probe unit for analyte nucleic acid recognition having a base sequence specific to an analyte nucleic acid; and plural tandemly ligated labeled probe units having mutually different base sequences and each comprising a fluorophore labeled internal base, wherein the fluorophore-labeled internal bases are placed at a distance of eight or more bases.