Patent US4265443A successfully refreshed from Google Patents.
Patent US4647529A successfully refreshed from Google Patents.
US4647529A
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US
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Hybridization method of detecting nucleic acid sequences with probe containing thionucleotide
Assignee: Individual
•
Filing: 1984-06-01
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Granted: 1987-03-03
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Expires: 2004-06-01
Abstract
Abstract Improved hybridization probe compositions and method for detecting nucleic acids are provided. A hybridization probe constructed to contain thionucleotides is hybridized to a nucleotide sequence of interest in the absence of formamide, DTT and dextran sulfate. If a cold ( 32 S) thionucleotide is incorporated into the probe molecule, the probe is provided with a detectable label, radioactive or otherwise.
Inventors
- 👤 Karin D. Rodland
- 👤 Peter J. Russell
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 13:53
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6888; C12Q1/6895; C07; C07H; C07H21/00; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 55
Claim 1
Independent Claim
73 words
A method for detecting the presence of a nucleotide sequence in substantially single stranded form in a sample suspected of containing said sequence, said method comprising: annealing to said nucleotide sequence, a probe that is complementary to said suspected sequence and contains both a detectable label other than 35 S and a thionucleotide in which the thiol group is non-radioactive; and detecting the presence of the labeled probe annealed to said nucleotide sequence.
Claim 2
Independent Claim
73 words
A method for detecting the presence of a nucleotide sequence in substantially single stranded form in a sample suspected of containing said sequence, said method comprising: annealing to said nucleotide sequence, a probe that is complementary to said suspected sequence and contains both a detectable label other than 35 S and a thionucleotide in which the thiol group is non-radioactive; and detecting the presence of the labeled probe annealed to said nucleotide sequence.
Claim 3
Dependent Claim
21 words
A method according to claim 1 wherein said non-radioactive thionucleotide is either deoxycytosine 5'αthio-triphosphateα-[ 32 S] or deoxyadenosine 5'αthio-triphosphateα-[ 32 S].
Claim 4
Dependent Claim
16 words
A method according to claim 2 wherein said probe has a radioactive isotope as a label.
Claim 5
Dependent Claim
23 words
A method according to claim 3 wherein said radioactive isotope is one of 3 H, 14 C, 32 P, or I 125 .
Claim 6
Dependent Claim
21 words
A method according to claim 2 wherein said probe is labeled with both a radioactive isotope and a detectable chemical modification.
Claim 7
Dependent Claim
31 words
A method according to claim 5 wherein said chemical modification is a biotinylated nucleotide and said radioactive isotope is one of 3 H, 14 C, 32 P, or I 125 .
Claim 8
Dependent Claim
16 words
A method according to claim 2 wherein said probe is labeled with a detectable chemical modification.
Claim 9
Dependent Claim
21 words
A method according to claim 2 wherein said nucleotide sequence is immobilized on a membrane before being annealed to said probe.
Claim 10
Dependent Claim
14 words
A method according to claim 8 wherein said membrane is a nylon base membrane.
Claim 11
Dependent Claim
17 words
A method according to claim 8 wherein said membrane has a pore size greater than 0.45 microns.
Claim 12
Dependent Claim
21 words
A method according to claim 8 wherein said probe and nucleotide sequence are annealed in the absence of a nonpolar solvent.
Claim 13
Dependent Claim
15 words
A method according to claim 11 wherein the annealing occurs in the absence of formamide.
Claim 14
Dependent Claim
21 words
A method according to claim 8 wherein said probe and nucleotide sequence are annealed in the absence of a reducing agent.
Claim 15
Dependent Claim
23 words
A method according to claim 8 wherein said probe and nucleotide sequence are annealed in the absence of a sulfhydryl containing reducing agent.
Claim 16
Dependent Claim
18 words
A method according to claim 14 wherein said annealing occurs in the absence of either dithiothreitol or 2-mercaptoethanol.
Claim 17
Dependent Claim
20 words
A method according to claim 8 wherein said probe and nucleotide sequence are annealed in the absence of dextran sulfate.
Claim 18
Dependent Claim
26 words
A method according to claim 8 wherein said probe and nucleotide sequence are annealed in the absence of a nonpolar solvent, reducing agent and dextran sulfate.
Claim 19
Dependent Claim
19 words
A method according to claim 17 wherein said annealing occurs in the absence of dextran sulfate, formamide and dithiothreitol.
Claim 20
Dependent Claim
16 words
A method according to claim 2 wherein said nucleotide sequence is genomic DNA from Neurospora crassa.
Claim 21
Dependent Claim
14 words
A method according to claim 2 wherein said nucleotide sequence is human genomic DNA.
Claim 22
Dependent Claim
13 words
A method according to claim 2 wherein said nucleotide sequence is plasmid DNA.
Claim 23
Independent Claim
55 words
A method of constructing a probe for hybridizing to a nucleotide sequence of interest comprising: selecting a nucleic acid polymer complementary to said nucleotide sequence; substituting at least one thionucleotide having a non-radioactive thiol group for an analogous nucleotide present in said polymer; and labeling said polymer with a detectable label other than 35 S.
Claim 24
Independent Claim
55 words
A method of constructing a probe for hybridizing to a nucleotide sequence of interest comprising: selecting a nucleic acid polymer complementary to said nucleotide sequence; substituting at least one thionucleotide having a non-radioactive thiol group for an analogous nucleotide present in said polymer; and labeling said polymer with a detectable label other than 35 S.
Claim 25
Dependent Claim
21 words
A method according to claim 22 wherein said non-radioactive nucleotide is either deoxycytosine 5'αthio-triphosphateα-[ 32 S] or deoxyadenosine 5'αthio-triphosphateα-[ 32 S].
Claim 26
Dependent Claim
13 words
A method according to claim 23 wherein said label is a radioactive isotope.
Claim 27
Dependent Claim
22 words
A method according to claim 24 wherein said radioactive isotope is one of 3 H, 14 C, 32 P, or 125 I.
Claim 28
Dependent Claim
14 words
A method according to claim 22 wherein said label is a detectable chemical modification.
Claim 29
Dependent Claim
20 words
A method according to claim 22 wherein said probe is labeled with both a radioactive isotope and detectable chemical modification.
Claim 30
Dependent Claim
31 words
A hybridization probe of the formula ##STR6## wherein R is any purine or pyrimidine base, S is 32 S, and the probe has incorporated a radioactive isotope other than 35 S.
Claim 31
Dependent Claim
31 words
A hybridization probe of the formula ##STR6## wherein R is any purine or pyrimidine base, S is 32 S, and the probe has incorporated a radioactive isotope other than 35 S.
Claim 32
Dependent Claim
45 words
A probe according to claim 28 wherein said radioactive isotope is one of 3 H substituting for any H, 14 C substituting for any C, 32 P substituting for any P, or 125 I present in any iodinated modification in a purine or pyrimidine base.
Claim 33
Dependent Claim
17 words
A probe according to claim 28 wherein said purine or pyrimidine bases contains a detectable chemical modification.
Claim 34
Dependent Claim
25 words
A hybridization probe of the formula ##STR7## wherein R is any purine or pyrimidine base containing a detectable chemical modification and S is 32 S.
Claim 35
Dependent Claim
25 words
A hybridization probe of the formula ##STR7## wherein R is any purine or pyrimidine base containing a detectable chemical modification and S is 32 S.
Claim 36
Independent Claim
84 words
A method for detecting the presence of a nucleotide sequence in substantially single stranded DNA form in a sample suspected of containing said sequence, said method comprising: forming a labeled DNA probe that is complementary to said suspected sequence and contains a thionucleotide attached to a phosphate base of said probe; annealing said nucleotide sequence in the absence of a nonpolar solvent, reducing agent and dextran sulfate to said labeled probe; and detecting the presence of the labeled probe annealed to said nucleotide sequence.
Claim 37
Independent Claim
84 words
A method for detecting the presence of a nucleotide sequence in substantially single stranded DNA form in a sample suspected of containing said sequence, said method comprising: forming a labeled DNA probe that is complementary to said suspected sequence and contains a thionucleotide attached to a phosphate base of said probe; annealing said nucleotide sequence in the absence of a nonpolar solvent, reducing agent and dextran sulfate to said labeled probe; and detecting the presence of the labeled probe annealed to said nucleotide sequence.
Claim 38
Dependent Claim
20 words
A method according to claim 32 wherein the annealing occurs in the absence of a sulfhydryl-containing reducing agent and formamide
Claim 39
Dependent Claim
18 words
A method according to claim 33 wherein the annealing occurs in the absence of both dithiothreitol and 2-mercaptoethanol.
Claim 40
Independent Claim
79 words
A method for detecting the presence of a nucleotide sequence in substantially single stranded form in a sample suspected of containing said sequence, said method comprising: annealing to said nucleotide sequence, a probe that is complementary to said suspected sequence and contains an incorporated thionucleotide and an incorporated detectable label other than 35 S in addition to said thionucleotide; and detecting the presence of the probe annealed to said nucleotide sequence by detecting the signal generated by said label.
Claim 41
Independent Claim
79 words
A method for detecting the presence of a nucleotide sequence in substantially single stranded form in a sample suspected of containing said sequence, said method comprising: annealing to said nucleotide sequence, a probe that is complementary to said suspected sequence and contains an incorporated thionucleotide and an incorporated detectable label other than 35 S in addition to said thionucleotide; and detecting the presence of the probe annealed to said nucleotide sequence by detecting the signal generated by said label.
Claim 42
Dependent Claim
13 words
A method according to claim 35 wherein said label is a radioactive isotope.
Claim 43
Dependent Claim
22 words
A method according to claim 36 wherein said radioactive isotope is one of 3 H, 14 C, 32 P, or 125 I.
Claim 44
Dependent Claim
13 words
A method according to claim 35 wherein said label is a chemical modification.
Claim 45
Dependent Claim
14 words
A method according to claim 38 wherein said chemical modification is a biotinylated nucleotide.
Claim 46
Independent Claim
55 words
A method of constructing a probe for hybridizing to a nucleotide sequence of interest comprising: selecting a nucleic acid polymer complementary to said nucleotide sequence; substituting at least one thionucleotide for an analogous nucleotide present in said polymer; and labeling said polymer with a detectable label other than 35 S in addition to said thionucleotide.
Claim 47
Independent Claim
55 words
A method of constructing a probe for hybridizing to a nucleotide sequence of interest comprising: selecting a nucleic acid polymer complementary to said nucleotide sequence; substituting at least one thionucleotide for an analogous nucleotide present in said polymer; and labeling said polymer with a detectable label other than 35 S in addition to said thionucleotide.
Claim 48
Dependent Claim
13 words
A method according to claim 40 wherein said label is a radioactive isotope.
Claim 49
Dependent Claim
12 words
A method of claim 40 wherein said label is a chemical modification.
Claim 50
Dependent Claim
34 words
A hybridization probe of the formula ##STR8## wherein R is any purine or pyrimidine base, S is 35 S or 32 S, and the probe has incorporated a radioactive isotope other than 35 S.
Claim 51
Dependent Claim
34 words
A hybridization probe of the formula ##STR8## wherein R is any purine or pyrimidine base, S is 35 S or 32 S, and the probe has incorporated a radioactive isotope other than 35 S.
Claim 52
Dependent Claim
28 words
A hybridization probe of the formula ##STR9## wherein R is any purine or pyrimidine base containing a detectable chemical modification and S is 32 S or 35 S.
Claim 53
Dependent Claim
28 words
A hybridization probe of the formula ##STR9## wherein R is any purine or pyrimidine base containing a detectable chemical modification and S is 32 S or 35 S.
Claim 54
Independent Claim
51 words
A method of constructing a probe for hybridizing to a nucleotide sequence of interest comprising: selecting a nucleic acid polymer complementary to said nucleotide sequence; substituting at least one thionucleotide having a non-radioactive thiol group for an analogous nucleotide present in said polymer; and labeling said polymer with a detectable label.
Claim 55
Independent Claim
51 words
A method of constructing a probe for hybridizing to a nucleotide sequence of interest comprising: selecting a nucleic acid polymer complementary to said nucleotide sequence; substituting at least one thionucleotide having a non-radioactive thiol group for an analogous nucleotide present in said polymer; and labeling said polymer with a detectable label.
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