Method for forming self-assembly substance using oligonucleotide synthesized by gene amplification reaction, self-assembly substance and method for detecting gene

• US 7,393,636 B2
• Filed: 10/30/2002
• Issued: 07/01/2008
• Est. Priority Date: 11/08/2001
• Status: Expired due to Fees

• Current Assignee: EIDIA Co., Ltd. (Sekisui)
• Original Assignee: Sanko Junyaku Co., Ltd.
• Inventors: Hakii, Chikako, Usui, Mitsugu, Mitsuka, Mari
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Tung; Joyce
• Application Number:
  US10/495,065
• Publication Number:
  US 20060286553A1
• Time in Patent Office: 2,071 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides

Abstract

There is provided a method for forming a self-assembly substance using oligonucleotides without using special instruments or complicated procedures, an self-assembly substance formed by the method for forming the self-assembly substance, and a method for detecting an amplified target gene at a low cost and in a simple way by making use of the method for forming the self-assembly substance. In the method for forming the self-assembly substance using a self-assembly reaction of oligonucleotides, the oligonucleotides comprise oligonucleotides synthesized by a gene amplification reaction. The oligonucleotides synthesized by the gene amplification reaction are detected by forming a self-assembly substance by the use of the method for forming the self-assembly substance of oligonucleotides, and by detecting the formed self-assembly substance.

Claims

1. A method for forming a self-assembly substance of oligonucleotides, said method comprising:

(1) providing n (n≧

1) dimer-forming probe-bearing groups formed from a first group to a (2n−

1)th group in turn, wherein each group includes a plurality of pairs of dimer-forming probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 3 regions of a 3′

side region, a mid-region and a 5′

side region, in which the mid-regions of the oligonucleotides No. 1 and No. 2 have base sequences complementary to each other, and the 3′

side regions and 5′

side regions thereof have base sequences not complementary to each other;

(2) providing n (n≧

1) cross-linking probe-bearing groups formed from a second group to a 2 nth group in turn, wherein each group includes a plurality of pairs of cross-linking probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 2 regions of a 3′

side region and a 5′

side region, in which the 3′

side regions and the 5′

side regions of the oligonucleotides No. 1 and No. 2 have base sequences not complementary to each other, and the cross-linking probes having base sequences capable of cross-linking dimers formed from the dimer-forming probes; and

(3) hybridizing the dimer-forming probes of the dimer-forming probe-bearing groups with the cross-linking probes of the cross-linking probe-bearing groups, wherein the oligonucleotides are self-assembled to form the self-assembly substance of oligonucleotides,

wherein in the case of n=1, base sequences of the probes are made complementary to each other in the following respective pairs;

(a) the 3′

side region of the oligonucleotide No. 1 of the first group of the dimer-forming probe-bearing groups and the 3′

side region of the oligonucleotide No. 1 of the second group of the cross-linking probe-bearing groups;

(b) the 5′

side region of the oligonucleotide No. 2 of the first group of the dimer-forming probe-bearing groups and the 5′

side region of the oligonucleotide No. 2 of the second group of the cross-linking probe-bearing groups;

(c) the 3′

side region of the oligonucleotide No. 2 of the second group of the cross-linking probe-bearing groups and the 3′

side region of the oligonucleotide No. 2 of the first group of the dimer-forming probe-bearing groups; and

(d) the 5′

side region of the oligonucleotide No. 1 of the second group of the cross-linking probe-bearing groups and the 5′

side region of the oligonucleotide No. 1 of the first group of the dimer-forming probe-bearing groups.

5. The method for forming a self-assembly substance of oligonucleotides according to any one of claims 1-4, wherein at least one of the dimer-forming probes and the cross-linking probes is an oligonucleotide synthesized by a gene amplification reaction.

6. The method for forming a self-assembly substance of oligonucleotides according to any one of claims 1-4, wherein the hybridizing of the dimer-forming probes of the dimer-forming probe-bearing groups with the cross-linking probes of the cross-linking probe-bearing groups comprises:

forming dimers from the dimer-forming probes of the dimer-forming probe-bearing groups; and

hybridizing the dimers with the cross-linking probes of the cross-linking probe-bearing groups.

7. The method for forming a self-assembly substance of oligonucleotides according to any one of claims 1-4, wherein the pairs of the dimer-forming probes comprise plural kinds of pairs of dimer-forming probes differing in the mid-regions.

8. The method for forming a self-assembly substance of oligonucleotides according to any one of claims 1-4, wherein the pairs of the dimer-forming probes are provided with the same base sequences at the 3′

side regions and/or the 5′

side regions thereof.

9. The method for forming a self-assembly substance of oligonucleotides according to any one of claims 5, wherein at least one of the cross-linking probes is an oligonucleotide which comprises the 2 regions of the cross-linking probes and is synthesized by the gene amplification reaction.

10. The method for forming a self-assembly substance of oligonucleotides according to claim 9, wherein the oligonucleotides synthesized by the gene amplification reaction are gene fragments complementary to each other, and the gene fragments comprise at least 4 regions and include 2 regions complementary to the 5′

side region and the 3′

side region of the oligonucleotides of the (2n−

1)th group, respectively.

11. The method for forming a self-assembly substance of oligonucleotides according to any one of claims 1-4, wherein the dimer-forming probes and the cross-linking probes are comprised of at least one base selected from the group consisting of DNA, RNA, PNA and LNA.

12. The method for forming a self-assembly substance of oligonucleotides according to any one of claims 1-4, wherein at least one G (guanine) or C (cytosine) is arranged at one or more ends of the complementary base sequence regions of the dimer-forming probes and the cross-linking probes, and when hybridizing the probes, at least one G-C bond is formed at the ends of the complementary base sequence regions.

2. A method for forming a self-assembly substance of oligonucleotides, said method comprising:

(1) providing n (n≧

1) dimer-forming probe-bearing groups formed from a first group to a (2n−

1)th group in turn, wherein each group includes a plurality of pairs of dimer-forming probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 3 regions of a 3′

side region, a mid-region and a 5′

side region, in which the mid-regions of the oligonucleotides No. 1 and No. 2 have base sequences complementary to each other, and the 3′

side regions and 5′

side regions thereof have base sequences not complementary to each other;

(2) providing n (n≧

1) cross-linking probe-bearing groups formed from a second group to a 2 nth group in turn, wherein each group includes a plurality of pairs of cross-linking probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 2 regions of a 3′

side region and a 5′

side region, in which the 3′

side regions and the 5′

side regions of the oligonucleotides No. 1 and No. 2 have base sequences not complementary to each other, and the cross-linking probes having base sequences capable of cross-linking dimers formed from the dimer-forming probes; and

(3) hybridizing the dimer-forming probes of the dimer-forming probe-bearing groups with the cross-linking probes of the cross-linking probe-bearing groups, wherein the oligonucleotides are self-assembled to form the self-assembly substance of oligonucleotides,

wherein in the case of n=1, base sequences of the probes are made complementary to each other in the following respective pairs;

(a) the 3′

side region of the oligonucleotide No. 1 of the first group of the dimer-forming probe-bearing groups and the 3′

side region of the oligonucleotide No. 1 of the second group of the cross-linking probe-bearing groups;

(b) the 5′

side region of the oligonucleotide No. 2 of the first group of the dimer-forming probe-bearing groups and the 5′

side region of the oligonucleotide No. 1 of the second group of the cross-linking probe-bearing groups;

(c) the 3′

side region of the oligonucleotide No. 2 of the first group of the dimer-forming probe-bearing groups and the 3′

side region of the oligonucleotide No. 2 of the second group of the cross-linking probe-bearing groups; and

(d) the 5′

side region of the oligonucleotide No. 1 of the first group of the dimer-forming probe-bearing groups and the 5′

side region of the oligonucleotide No. 2 of the second group of the cross-linking probe-bearing groups.

3. A method for forming a self-assembly substance of oligonucleotides, said method comprising:

(1) providing n (n≧

1) dimer-forming probe-bearing groups formed from a first group to a (2n−

1)th group in turn, wherein each group includes a plurality of pairs of dimer-forming probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 3 regions of a 3′

side region, a mid-region and a 5′

side region, in which the mid-regions of the oligonucleotides No. 1 and No. 2 have base sequences complementary to each other, and the 3′

side regions and 5′

side regions thereof have base sequences not complementary to each other;

(2) providing n (n≧

1) cross-linking probe-bearing groups formed from a second group to a 2 nth group in turn, wherein each group includes a plurality of pairs of cross-linking probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 2 regions of a 3′

side region and a 5′

side region, in which the 3′

side regions and the 5′

side regions of the oligonucleotides No. 1 and No. 2 have base sequences not complementary to each other, and the cross-linking probes having base sequences capable of cross-linking dimers formed from the dimer-forming probes; and

(3) hybridizing the dimer-forming probes of the dimer-forming probe-bearing groups with the cross-linking probes of the cross-linking probe-bearing groups, wherein the oligonucleotides are self-assembled to form the self-assembly substance of oligonucleotides,

wherein in the case of n≧

2 base sequences of the probes are made complementary to each other in the following respective pairs;

(a) the 3′

side region of the oligonucleotide No. 1 of the (2n−

3)th group of the dimer-forming probe-bearing groups and the 3′

side region of the oligonucleotide No. 1 of the (2n−

2)th group of the cross-linking probe-bearing groups;

(b) the 5′

side region of the oligonucleotide No. 2 of the (2n−

3)th group of the dimer-forming probe-bearing groups and the 5′

side region of the oligonucleotide No. 2 of the (2n−

2)th group of the cross-linking probe-bearing groups;

(c) the 3′

side region of the oligonucleotide No. 2 of the (2n−

2)th group of the cross-linking probe-bearing groups and the 3′

side region of the oligonucleotide No. 2 of the (2n−

1)th group of the dimer-forming probe-bearing groups;

(d) the 5′

side region of the oligonucleotide No. 1 of the (2n−

2)th group of the cross-linking probe-bearing groups and the 5′

side region of the oligonucleotide No. 1 of the (2n−

1)th group of the dimer-forming probe-bearing groups;

(e) the 3′

side region of the oligonucleotide No. 1 of the last group of the dimer-forming probe-bearing groups and the 3′

side region of the oligonucleotide No. 1 of the last group of the cross-linking probe-bearing groups;

(f) the 5′

side region of the oligonucleotide No. 2 of the last group of the dimer-forming probe-bearing groups and the 5′

side region of the oligonucleotide No. 2 of the last group of the cross-linking probe-bearing groups;

(g) the 3′

side region of the oligonucleotide No. 2 of the last group of the cross-linking probe-bearing groups and the 3′

side region of the oligonucleotide No. 2 of the first group of the dimer-forming probe-bearing groups; and

(h) the 5′

side region of the oligonucleotide No. 1 of the last group of the cross-linking probe-bearing groups and the 5′

side region of the oligonucleotide No. 1 of the first group of the dimer-forming probe-bearing groups.

4. A method for forming a self-assembly substance of oligonucleotides, said method comprising:

(1) providing n (n≧

1) dimer-forming probe-bearing groups formed from a first group to a (2n−

1)th group in turn, wherein each group includes a plurality of pairs of dimer-forming probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 3 regions of a 3′

side region, a mid-region and a 5′

side region, in which the mid-regions of the oligonucleotides No. 1 and No. 2 have base sequences complementary to each other, and the 3′

side regions and 5′

side regions thereof have base sequences not complementary to each other;

(2) providing n (n≧

1) cross-linking probe-bearing groups formed from a second group to a 2 nth group in turn, wherein each group includes a plurality of pairs of cross-linking probes composed of a pair of an oligonucleotide No. 1 and an oligonucleotide No. 2, each oligonucleotide having 2 regions of a 3′

side region and a 5′

side region, in which the 3′

side regions and the 5′

side regions of the oligonucleotides No. 1 and No. 2 have base sequences not complementary to each other, and the cross-linking probes having base sequences capable of cross-linking dimers formed from the dimer-forming probes; and

(3) hybridizing the dimer-forming probes of the dimer-forming probe-bearing groups with the cross-linking probes of the cross-linking probe-bearing groups, wherein the oligonucleotides are self-assembled to form the self-assembly substance of oligonucleotides,

wherein in the case of n≧

2 base sequences of the probes are made complementary to each other in the following respective pairs;

(a) the 3′

side region of the oligonucleotide No. 1 of the (2n−

3)th group of the dimer-forming probe-bearing groups and the 3′

side region of the oligonucleotide No. 1 of the (2n−

2)th group of the cross-linking probe-bearing groups;

(b) the 5′

side region of the oligonucleotide No. 2 of the (2n−

3)th group of the dimer-forming probe-bearing groups and the 5′

side region of the oligonucleotide No. 2 of the (2n−

2)th group of the cross-linking probe-bearing groups;

(c) the 3′

side region of the oligonucleotide No. 2 of the (2n−

2)th group of the cross-linking probe-bearing groups and the 3′

side region of the oligonucleotide No. 2 of the (2n−

1)th group of the dimer-forming probe-bearing groups;

(d) the 5′

side region of the oligonucleotide No. 1 of the (2n−

2)th group of the cross-linking probe-bearing groups and the 5′

side region of the oligonucleotide No. 1 of the (2n−

1)th group of the dimer-forming probe-bearing groups;

(e) the 3′

side region of the oligonucleotide No. 1 of the last group of the dimer-forming probe-bearing groups and the 3′

side region of the oligonucleotide No. 1 of the last group of the cross-linking probe-bearing groups;

(f) the 5′

side region of the oligonucleotide No. 2 of the last group of the dimer-forming probe-bearing groups and the 5′

side region of the oligonucleotide No. 1 of the last group of the cross-linking probe-bearing groups;

(g) the 3′

side region of the oligonucleotide No. 2 of the last group of the cross-linking probe-bearing groups and the 3′

side region of the oligonucleotide No. 2 of the first group of the dimer-forming probe-bearing groups; and

(h) the 5′

side region of the oligonucleotide No. 2 of the last group of the cross-linking probe-bearing groups and the 5′

side region of the oligonucleotide No. 1 of the first group of the dimer-forming probe-bearing groups.