Probe for constructing probe-polymer method of constructing probe-polymer and utilization thereof
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.
1. Probes for forming a probe-polymer comprising a first probe and a second probe having the following characteristics (a), (b), (c), and (d):
(a) the first and second probes each comprising n base sequence regions complementary to each other, wherein 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 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, wherein n is at least 3;
(b) when the first and second probes are reacted with each other, the X 1 region hybridizes only to the X 1 ′ region, the X 2 region hybridizes only to the X 2 ′ region, the X 3 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 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 both terminals of each one of the complementary base sequence regions in the first and second probes, and upon hybridization of the first and second probes, at least one C—G bond is formed at all of the terminals of the complementary regions; and
(d) the number of bases in each complementary base sequence region in the first and second probes is at least 8.
2. The probes for forming a probe-polymer according to claim 1 , wherein the number (n) of complementary base sequence regions in the first and second probes is 3, 4, 5, or 6.
3. The probes for forming a probe-polymer according to claim 1 or 2 , wherein the first and second probes comprise bases selected from DNA, RNA, or PNA.
4. A reagent for detecting a target gene in a sample, comprising a first probe and a second probe as polymerization probes having the following characteristics (a), (b), (c), (d), and (e) as essential elements:
(a) the first and second probes each comprising n base sequence regions complementary to each other, wherein 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 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, wherein n is at least 3;
(b) when the first and second probes are reacted with each other, the X 1 region hybridizes only to the X 1 ′ region, the X 2 region hybridizes only to the X 2 ′ region, the X 3 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 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 both terminals of each one of the complementary base sequence regions in the first and second probes, and upon hybridization of the first and second probes, at least one C—G bond is formed at all of the terminals of the complementary regions;
(d) one of the 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; and
(e) the number of bases in each complementary base sequence region in the first and second probes is at least 8.
5. A reagent for detecting a target gene in a sample, comprising: a first and a second probe having the following characteristics (a), (b), (c), and (d) as polymerization probes; and at least one target gene capture probe comprising 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 essential elements,
(a) the first and second probes each comprising n base sequence regions complementary to each other, wherein 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 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, wherein n is at least 3;
(b) when the first and second probes are reacted with each other, the X 1 region hybridizes only to the X 1 ′ region, the X 2 region hybridizes only to the X 2 ′ region, the X 3 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 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 both terminals of each one of the complementary base sequence regions in the first and second probes, and upon hybridization of the first and second probes, at least one C—G bond is formed at all of the terminals of the complementary regions; and
(d) the number of bases in each complementary base sequence region in the first and second probes is at least 8.
6. The reagent for detecting a target gene according to claim 4 or 5 , wherein the number (n) of complementary base sequence regions in the first and second probes is 3, 4, 5, or 6.
7. The reagent for detecting a target gene according to claim 4 or 5 , wherein the first and second probes comprise bases selected from DNA, RNA, or PNA.