METHOD OF DETECTING NUCLEOTIDE SEQUENCE WITH AN INTRAMOLECULAR PROBE
First Claim
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.
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Accused Products
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.
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Citations
50 Claims
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1. A nucleotide sequence-detecting method, comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16)
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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.
- 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′
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17. A nucleotide mutation-analyzing method, comprising:
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(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. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 37, 38, 39, 40)
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35. A nucleotide mutation-analyzing method, comprising:
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(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, andthe 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. - View Dependent Claims (36)
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41. A nucleotide sequence-detecting method, comprising:
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(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). - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50)
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Specification