Probe for constructing probe polymer method of constructing probe polymer and utilization thereof
First Claim
Patent Images
1. Probe polymer forming probes comprising a pair of first and second probes having the following characteristics (a), (b) and (c):
- (a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the X′
n region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer; and
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions.
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Abstract
The present invention provides a method for measuring a target gene under isothermal conditions without using enzyme. A pair of probes each having n (n≧3) base sequence regions complementary to each other are hybridized alternately to form a double-stranded probe-polymer. A base pair at branched sites of each complementary base sequence region is designed to be a G (guanine)-C (cytosine) bond, whereby a stable double-stranded probe-polymer is formed. One of complementary portions in one probe is constituted to have a base sequence complementary to a part of a target gene, whereby a target gene-probe-polymer complex is formed and the target gene is measured.
26 Citations
26 Claims
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1. Probe polymer forming probes comprising a pair of first and second probes having the following characteristics (a), (b) and (c):
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(a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the X′
n region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer; and
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions. - View Dependent Claims (2, 3, 4)
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5. A method for forming probe-polymer by polymerizing a plurality of pairs of first and second probes having the following characteristics (a), (b) and (c):
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(a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the Xn region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer; and
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions. - View Dependent Claims (6, 7, 8)
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9. A probe-polymer obtained by polymerizing a plurality of pairs of first and second probes having the following characteristics (a), (b) and (c):
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(a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the Xn region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer; and
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions. - View Dependent Claims (10, 11, 12, 15, 16, 17, 20, 21, 22, 24, 25, 26)
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13. A method for measuring a target gene in a sample by the use of polymerization probes, comprising the following steps (1), (2) and (3):
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(1) with a pair of first and second probes having the following characteristics (a), (b) and (c) as polymerization probes, reacting either one of the probes having one base sequence region complementary to a part of a target gene with a sample so as to bind the probe to the target gene in the sample, (a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the Xn region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe hybridizes to such a region in the other probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer; and
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions;
(2) then reacting a plurality of the polymerization probes with each other to form a target gene-probe-polymer complex; and
(3) washing the unreacted probes off from the used polymerization probes, and measuring the amount of the formed probe-polymer.
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14. A method for measuring a target gene in a sample by the use of polymerization probes, comprising the following steps (1), (2) and (3):
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(1) with a pair of first and second probes having the following characteristics (a), (b) and (c) as polymerization probes, reacting at least one target gene capture probe with a sample so as to bind the capture probe to a target gene, the target gene capture probe being composed of two regions, one region of which is a base sequence region complementary to a part of the target gene and the other region of which is a base sequence region complementary to one region in either one of the two polymerization probes, (a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the Xn region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer; and
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions;
(2) then reacting the polymerization probes with each other so as to bind the capture probe to the polymerization probes to form a target gene-probe-polymer complex; and
(3) washing the unreacted probes off from the used polymerization probes and measuring the amount of the formed probe-polymer.
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18. A regent for detecting a target gene in a sample, comprising a pair of first and second probes as polymerization probes having the following characteristics (a), (b), (c) and (d) as essential elements:
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(a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the Xn region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer;
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions; and
(d) one of complementary base sequence regions in either one of the first or second probe has a region having a base sequence complementary to a part of the target gene.
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19. A reagent for detecting a target gene in a sample, comprising:
- a pair of first and second probes the following characteristics (a), (b) and (c) as polymerization probes; and
at least one target gene capture probe composed of at least two regions, one region of which is a base sequence region complementary to a part of the target gene and the other region of which is a base sequence region complementary to one region in either one of the two polymerization probes as the essential elements,(a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n region provided in this order from the 5′
-terminal of the second probe;
(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the Xn region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer; and
(c) at least one G (guanine) or C (cytosine) is arranged at branched sites of complementary base sequence regions in a pair of the first and second probes, and upon hybridization of a pair of the first and second probes, at least one C—
G bond is formed at the terminal of the complementary regions.
- a pair of first and second probes the following characteristics (a), (b) and (c) as polymerization probes; and
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23. Probe polymer forming probes comprising a pair of first and second probes having the following characteristics (a) and (b):
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(a) a pair of the first and second probes each composed of n (n≧
3) base sequence regions complementary to each other, wherein an X1 region, an X2 region, an X3 region, . . . an Xn region provided in this order from the 5′
-terminal of the first probe have base sequences complementary respectively to an X′
1 region, an X′
2 region, an X′
3 region, . . . an X′
n l region provided in this order from the 5′
-terminal of the second probe, and(b) when a pair of the first and second probes are reacted with each other, the X1 region hybridizes only to the X′
1 region, the X2 region hybridizes only to the X′
2 region, the X3 region hybridizes only to the X′
3 region, . . . and the Xn region hybridizes only to the X′
n region, and when both the probes are bound, they hybridize to each other at any one of the regions in one probe, and a plurality of the pairs of the first and second probes bound at the one region hybridize to each other to form a probe-polymer.
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Specification
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Current AssigneeEIDIA Co., Ltd. (Sekisui)
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Original AssigneeSanko Junyaku Co., Ltd.
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InventorsUsui, Mitsugu, Hakii, Chikako, Mitsuka, Mari
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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current435/6
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CPC Class CodesC12N 15/10 Processes for the isolation...C12Q 1/6811 Selection methods for produ...C12Q 1/6816 characterised by the detect...C12Q 1/682 Signal amplificationC12Q 2525/161 incorporating target specif...C12Q 2525/185 incorporating bases where t...C12Q 2525/313 Branched oligonucleotidesC12Q 2527/101 TemperatureC12Q 2537/143 Multiplexing, i.e. use of m...C12Q 2563/179 the label being a nucleic acid
