Method of detecting nucleotide sequence with an intramolecular probe
First Claim
1. A nucleotide sequence-detecting method, comprising:
- (a) preparing a nucleic acid sample;
(b) preparing a first intramolecular detecting sequence having a sequence complementary to a first sequence located on a 3′
-side of the detecting site contained in the nucleic acid sample and a second intramolecular detecting sequence having a sequence complementary to a second sequence located on 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 nucleic acid 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 a desired sequence in the nucleic acid sample from the cyclic structure.
2 Assignments
0 Petitions
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.
18 Citations
42 Claims
-
1. A nucleotide sequence-detecting method, comprising:
-
(a) preparing a nucleic acid sample; (b) preparing a first intramolecular detecting sequence having a sequence complementary to a first sequence located on a 3′
-side of the detecting site contained in the nucleic acid sample and a second intramolecular detecting sequence having a sequence complementary to a second sequence located on 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 nucleic acid 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 a desired sequence in the nucleic acid sample from the cyclic structure. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
-
-
14. A nucleotide mutation-analyzing method, comprising:
-
(a) preparing a duplicated chain having a sequence that is the same as a nucleic acid analyte having a mutation site and connecting a first and a second fragment to 3′ and
5′
terminals of the duplicated chain to obtain a detecting chain, the first fragment having a sequence complementary to the mutation site and the region around the mutation site on the nucleic acid analyte the duplicated chain or to the region around the mutation site on the nucleic acid analyte,wherein the sequences of the first fragment and the second fragment are different from each other, and the sequence of the first fragment or the second fragment includes a sequence complementary to the sequence of the mutation site on the nucleic acid analyte; (b) making hybridizations at two regions on the detecting chain to form a dumbbell structure, wherein one region is between the mutation site and the region around the mutation site on the detecting chain and the first fragments in the detecting chain, another region is between the region around the mutation site on the detecting chain and the second fragments in the detecting chain; (c) connecting the terminals of the detecting chain in the form of dumbbell structure to each other, to form a closed ring-detecting chain, if a desired sequence is present on the mutation site to be detected, through a nucleic acid monomer or a nucleic acid complementary to that of the mutation site or directly; (d) preparing a fragment having a sequence containing the connecting region of the closed ring-detecting chain or its complementary chain sequence, or both of them; and (e) analyzing a nucleotide mutation by detecting presence of the sequence containing the connecting region of the prepared closed ring-detecting chain or its complementary sequence. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
-
-
32. A nucleotide mutation-analyzing method, comprising:
-
(a) carrying out amplification for a nucleic acid analyte using primers to obtain an amplification product as duplicated chains containing a first detecting chain having the same sequence as that of the nucleic acid analyte and a second detecting chain complementary to the first single-stranded chain, wherein said primers include first and second primers for amplification of a sequence containing the mutation sequence of the nucleic acid analyte, the first primer includes a first priming sequence and a first probe sequence, the first priming sequence located on a 3′
terminal side of the first primer, and the first probe sequence located on a 5′
terminal side of the first primer, and the second primer includes a second priming sequence and a second probe sequence, the second priming located on a 3′
terminal side of the second primer and the second probe sequence on a 5′
terminal side of the second primer,the first probe sequence consists of a sequence which is the same as that of the mutation side and a further sequence which is the same as that of sequence located between the 3′
side of the mutation side and the 5′
side of a site for binding of the first priming sequence on the nucleic acid analyte, the second probe sequence is complementary to a sequence located at between 5′
side of the mutation site on the nucleic acid analyte and 3′
side of a site for binding of the second priming sequence on the nucleic acid analyte, and the 5′
terminal of the second primer is phosphorylated;(b) converting the amplification product obtained in step (a) into the first detecting chain and the second detecting chain; (c) making the first detecting chain and/or the second detecting chain hybridize intramolecularly to form a dumbbell structure, and if a desired sequence is present on the mutation site to be detected, from the dumbbell structure by ring-closure reaction to give a closed ring-detecting chain; and (d) the mutation to be detected in the nucleic acid analyte is analyzed by detecting the difference in conformation between the closed ring-detecting chain and un-closed first and/or second detecting chain. - View Dependent Claims (33)
-
-
34. A nucleotide sequence-detecting method, comprising:
-
(a) preparing a nucleic acid sample, wherein the nucleic acid sample contains a detecting site, a first sequence located on a 3′
side of the detecting site, and a second sequence located on a 5′
side of the detecting site;(b) preparing a first detecting chain-preparing nucleic acid and a second detecting chain-preparing nucleic acid, wherein the first detecting chain-preparing nucleic acid contains a first probe sequence located on a 5′
terminal side of the first detecting chain-preparing nucleic acid and a primer sequence located on a 3′
terminal side of the first detecting chain-preparing nucleic acid, the first probe sequence being same sequence as that of the first sequence of the nucleic acid sample, the primer sequence complementary to that of a region located in a 3′
side of the first sequence of the nucleic acid sample,the second detecting chain-preparing nucleic acid contains a second probe sequence located on a 3′
terminal side of the second detecting chain-preparing nucleic acid and a oligonucleotide sequence on a 5′
terminal side of the second detecting chain-preparing nucleic acid, a 3′
terminal of the second probe sequence having a complementary chain synthesis-inhibiting structure,the second probe sequence being same sequence as that of the second sequence on the nucleic acid sample, the oligonucleotide sequence complementary to that of a region located in a 5′
-side of the second sequence of nucleic acid sample,wherein at least one of the 5′
-terminal nucleotide of the first detecting chain-preparing nucleic acid and the 3′
-terminal nucleotide of the second detecting chain-preparing nucleic acid is modified in such a manner that they can bind to each other;(c) preparing a detecting chain by allowing the primer sequence and the oligonucleotide sequence to hybridize to the nucleic acid sample, allowing elongation reaction of the first detecting chain-preparing nucleic acid with initiation from a 3′
terminal of the primer sequence, and allowing ligation reaction between a 3′
terminal of the elongated first detecting chain preparing nucleic acid and a 5′
terminal of the second detecting chain-preparing nucleic acid;(d) allowing intramolecular hybridization of the detecting chain at two positions, one position being between a site corresponding to the first sequence and the first probe sequence on the detecting chain, and another position being between a site corresponding to the second sequence and the second probe sequence on the detecting chain detecting chain preparing nucleotides to form a dumbbell structure; (e) forming a closed ring-detecting chain by connecting between 3′ and
5′
terminals of the detecting chain in the form of dumbbell structure;(f) amplifying a nucleic acid having the sequence containing the connecting region of the closed ring-detecting chain; and (g) detecting the detecting-site sequence in the nucleic acid sample by detecting the amplification product obtained by amplification (f). - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42)
-
Specification