US5185243A
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Method for detection of specific nucleic acid sequences
Assignee: Siemens Healthcare Diagnostics GmbH Germany
•
Filing: 1988-08-25
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Granted: 1993-02-09
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Expires: 2008-08-25
Abstract
Abstract A method is disclosed for detecting the presence of a target nucleotide sequence in a polynucleotide. The method comprises hybridizing a first nucleotide sequence and a second nucleotide sequence to non-contiguous portions of a target nucleotide sequence, covalently attaching the first and second sequences when they are hybridized to the target sequence, and determining the presence of covalently attached first and second sequences. The presence of the covalently attached first and second sequences is related to the presence of the target nucleotide sequence. The invention may be applied to target nucleotide sequences in DNA or RNA. Specific target nucleotide sequences of interest will frequently be characteristic of particular microorganisms, viruses, viroids, or genetic characteristics, including genetic abnormalities.
Inventors
- 👤 Edwin F. Ullman
- 👤 Thomas C. Goodman
- 👤 Paul D. Stull
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 19:37
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6834; C12Q1/6876; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/975; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 66
Claim 1
Independent Claim
66 words
A method for detecting the presence of a target nucleotide sequence, which comprises: hydridizing a first nucleotide sequence and a second nucleotide sequence to non-contiguous portions of a target nucleotide sequence, covalently attaching said first and second sequences when hybridized to said target sequence, and determining the presence of covalently attached first and second sequences, the presence thereof confirming the presence of said target nucleotide sequence.
Claim 2
Independent Claim
66 words
A method for detecting the presence of a target nucleotide sequence, which comprises: hydridizing a first nucleotide sequence and a second nucleotide sequence to non-contiguous portions of a target nucleotide sequence, covalently attaching said first and second sequences when hybridized to said target sequence, and determining the presence of covalently attached first and second sequences, the presence thereof confirming the presence of said target nucleotide sequence.
Claim 3
Independent Claim
143 words
A method for detecting a target nucleotide sequence in a polynucleotide, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence, (b) a first polynucleotide having a sequence complementary to a first portion of said target nucleotide sequence, (c) a second polynucleotide having a sequence complementary to a second portion of said target nucleotide sequence other than and non-contiguous with said first portion, and (d) means for covalently attaching said first and second polynucleotides when said polynucleotides are hybridized with said target nucleotide sequence, under conditions wherein said first and second polynucleotides hybridize with said target nucleotide sequence and become covalently attached when said target nucleotide sequence is present; and determining the presence of covalently attached first and second polynucleotides, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
Claim 4
Independent Claim
143 words
A method for detecting a target nucleotide sequence in a polynucleotide, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence, (b) a first polynucleotide having a sequence complementary to a first portion of said target nucleotide sequence, (c) a second polynucleotide having a sequence complementary to a second portion of said target nucleotide sequence other than and non-contiguous with said first portion, and (d) means for covalently attaching said first and second polynucleotides when said polynucleotides are hybridized with said target nucleotide sequence, under conditions wherein said first and second polynucleotides hybridize with said target nucleotide sequence and become covalently attached when said target nucleotide sequence is present; and determining the presence of covalently attached first and second polynucleotides, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
Claim 5
Dependent Claim
12 words
The method of claim 2 wherein said target nucleotide sequence is DNA.
Claim 6
Dependent Claim
34 words
The method of claim 2 wherein said means for covalently attaching comprises means for extending said second polynucleotide when said sequence is hybridized with said target nucleotide sequence to render said complementary sequences contiguous.
Claim 7
Dependent Claim
20 words
The method of claim 4 wherein said means for extending comprises a polynucleotide polymerase and at least one nucleoside triphosphate.
Claim 8
Dependent Claim
17 words
The method of claim 5 wherein said nucleoside triphosphate is selected from dATP, dGTP, dCTP and dTTP.
Claim 9
Dependent Claim
31 words
The method of claim 2 wherein said first polynucleotide is at least 10 nucleotides in length and the combined length of said first and second polynucleotides is at least 20 nucleotides.
Claim 10
Dependent Claim
19 words
The method of claim 2 wherein said means for covalently attaching is an enzymatic means or a chemical means.
Claim 11
Dependent Claim
23 words
The method of claim 2 wherein said first or said second polynucleotides has, or is capable of having, means for immobilizing said polynucleotide.
Claim 12
Dependent Claim
13 words
The method of claim 9 wherein said means for immobilizing is a support.
Claim 13
Dependent Claim
21 words
The method of claim 2 wherein said first or said second polynucleotide has, or is capable of having, a reporter group.
Claim 14
Dependent Claim
29 words
The method of claim 11 wherein said reporter group is selected from the group consisting of biotin, fluorescers, chemiluminescers, radioactive groups, catalysts, enzymes, substrates, coenzymes, and amplifiable nucleotide sequences.
Claim 15
Independent Claim
171 words
A method for detecting a target nucleotide sequence, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence, (b) a first nucleotide sequence complementary to a first portion of said target nucleotide sequence, (c) a second nucleotide sequence complementary to a portion of said target nucleotide sequence other than and non-contiguous with said first portion, (d) means for extending said second nucleotide sequence when said sequence is hybridized with said target nucleotide sequence to render said first and second nucleotide sequences contiguous, and (e) means for covalently attaching said first and second sequences, under conditions wherein said first and second sequences hybridize with said target nucleotide sequence, said second sequence is extended to render said sequence contiguous with said first sequence and said first and second sequences become covalently attached when said target nucleotide sequence is present; and determining the presence of covalently attached first and second sequences, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
Claim 16
Independent Claim
171 words
A method for detecting a target nucleotide sequence, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence, (b) a first nucleotide sequence complementary to a first portion of said target nucleotide sequence, (c) a second nucleotide sequence complementary to a portion of said target nucleotide sequence other than and non-contiguous with said first portion, (d) means for extending said second nucleotide sequence when said sequence is hybridized with said target nucleotide sequence to render said first and second nucleotide sequences contiguous, and (e) means for covalently attaching said first and second sequences, under conditions wherein said first and second sequences hybridize with said target nucleotide sequence, said second sequence is extended to render said sequence contiguous with said first sequence and said first and second sequences become covalently attached when said target nucleotide sequence is present; and determining the presence of covalently attached first and second sequences, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
Claim 17
Dependent Claim
12 words
The method of claim 13 wherein said target nucleotide sequence is DNA.
Claim 18
Dependent Claim
20 words
The method of claim 13 wherein said means for extending comprises a polynucleotide polymerase and at least one nucleoside triphosphates.
Claim 19
Dependent Claim
17 words
The method of claim 15 wherein said nucleoside triphosphate is selected from dATP, dGTP, dCTP and dTTP.
Claim 20
Dependent Claim
19 words
The method of claim 16 wherein no more than three of said dATP, dGTP, dCTP and dTTP are used.
Claim 21
Dependent Claim
33 words
The method of claim 13 wherein said first nucleotide sequence is at least 10 nucleotides in length and the combined length of said first and second nucleotide sequences is at least 20 nucleotides.
Claim 22
Dependent Claim
19 words
The method of claim 13 wherein said means for covalently attaching is an enzymatic means or a chemical means.
Claim 23
Dependent Claim
24 words
The method of claim 13 wherein said first or said second nucleotide sequence has, or is capable of having, means for immobilizing said sequence.
Claim 24
Dependent Claim
13 words
The method of claim 20 wherein said means for immobilizing is a surface.
Claim 25
Dependent Claim
21 words
The method of claim 13 wherein said first or said second sequence has, or is capable of having, a reporter group.
Claim 26
Dependent Claim
28 words
The method of claim 22 wherein said reporter group is selected from the group consisting of fluorescers, chemiluminescers, radioactive groups, catalysts, enzymes, substrates, co-enzymes, and amplifiable nucleotide sequences.
Claim 27
Independent Claim
128 words
A method for detecting a target nucleotide sequence, which comprises: combining in a liquid medium (a) a sample suspected of containing said target nucleotide sequence, (b) a first nucleotide sequence capable of binding to a first portion of said target sequence, and (c) a second nucleotide sequence capable of binding to a second portion of said target sequence other than and non-contiguous with said first portion under conditions wherein said first and second nucleotide sequences hybridize with said target sequence; adding to said medium means for covalently attaching said first and second nucleotide sequences when said sequences are hybridized to said target sequence, and determining whether said first and second sequences have become covalently attached as an indication of the presence of said target sequence in said sample.
Claim 28
Independent Claim
128 words
A method for detecting a target nucleotide sequence, which comprises: combining in a liquid medium (a) a sample suspected of containing said target nucleotide sequence, (b) a first nucleotide sequence capable of binding to a first portion of said target sequence, and (c) a second nucleotide sequence capable of binding to a second portion of said target sequence other than and non-contiguous with said first portion under conditions wherein said first and second nucleotide sequences hybridize with said target sequence; adding to said medium means for covalently attaching said first and second nucleotide sequences when said sequences are hybridized to said target sequence, and determining whether said first and second sequences have become covalently attached as an indication of the presence of said target sequence in said sample.
Claim 29
Dependent Claim
12 words
The method of claim 24 wherein said target nucleotide sequence is DNA.
Claim 30
Dependent Claim
33 words
The method of claim 24 wherein said means for covalently attaching comprises means for extending said second nucleotide sequence when said sequence is hybridized to said target polynucleotide to render said sequences contiguous.
Claim 31
Dependent Claim
20 words
The method of claim 26 wherein said means for extending comprises a polynucleotide polymerase and at least one nucleoside triphosphate.
Claim 32
Dependent Claim
17 words
The method of claim 27 wherein said nucleoside triphosphate is selected from dATP, dGTP, dCTP and dTTP.
Claim 33
Dependent Claim
19 words
The method of claim 28 wherein no more than three of said dATP, dGTP, dCTP, and dTTP are used.
Claim 34
Dependent Claim
33 words
The method of claim 24 wherein said first nucleotide sequence is at least 10 nucleotides in length and the combined length of said first and second nucleotide sequences is at least 20 nucleotides.
Claim 35
Dependent Claim
19 words
The method of claim 24 wherein said means for covalently attaching is an enzymatic means or a chemical means.
Claim 36
Dependent Claim
26 words
The method of claim 24 wherein said first or said second nucleotide sequence has, or is capable of having, means for immobilizing said first nucleotide sequence.
Claim 37
Dependent Claim
13 words
The method of claim 32 wherein said means for immobilizing is a surface.
Claim 38
Dependent Claim
21 words
The method of claim 24 wherein said first or said second sequence has, or is capable of having, a reporter group.
Claim 39
Dependent Claim
28 words
The method of claim 34 wherein said reporter group is selected from the group consisting of fluorescers, chemiluminescers, radioactive groups, catalysts, enzymes, substrates, coenzymes, and amplifiable nucleotide sequences.
Claim 40
Independent Claim
191 words
A method for detecting a target nucleotide sequence, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence, (b) a first nucleotide sequence that is capable of binding to a portion of said target nucleotide sequence and has, or is capable of having, means for immobilizing said first polynucleotide, (c) a second nucleotide sequence that is capable of binding to a portion of said target nucleotide sequence other than the portion to which said first nucleotide sequence binds or to a portion contiguous thereto and that has, or is capable of having, a reporter group, and (d) means for causing said first nucleotide sequence to become covalently bound to said second nucleotide sequence; incubating said medium under conditions wherein said first and second nucleotide sequences hybridize with said target nucleotide sequence and become covalently bound to one another if said target nucleotide sequence is present; separating said first nucleotide sequence from said medium; and examining said separated first nucleotide sequence for the presence of said second nucleotide sequence, the presence thereof confirming the presence of said target nucleotide sequence in said sample.
Claim 41
Independent Claim
191 words
A method for detecting a target nucleotide sequence, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence, (b) a first nucleotide sequence that is capable of binding to a portion of said target nucleotide sequence and has, or is capable of having, means for immobilizing said first polynucleotide, (c) a second nucleotide sequence that is capable of binding to a portion of said target nucleotide sequence other than the portion to which said first nucleotide sequence binds or to a portion contiguous thereto and that has, or is capable of having, a reporter group, and (d) means for causing said first nucleotide sequence to become covalently bound to said second nucleotide sequence; incubating said medium under conditions wherein said first and second nucleotide sequences hybridize with said target nucleotide sequence and become covalently bound to one another if said target nucleotide sequence is present; separating said first nucleotide sequence from said medium; and examining said separated first nucleotide sequence for the presence of said second nucleotide sequence, the presence thereof confirming the presence of said target nucleotide sequence in said sample.
Claim 42
Dependent Claim
14 words
The method of claim 36 wherein said target nucleotide sequence in DNA or RNA.
Claim 43
Dependent Claim
35 words
The method of claim 36 wherein said means for causing comprises means for extending said first or said second nucleotide sequence when said sequences are hybridized to said target sequence to render said sequences contiguous.
Claim 44
Dependent Claim
20 words
The method of claim 38 wherein said means for extending comprises a polynucleotide polymerase and at least one nucleoside triphosphate.
Claim 45
Dependent Claim
17 words
The method of claim 39 wherein said deoxynucleoside triphosphate is selected from dATP, dGTP, dCTP and dTTP.
Claim 46
Dependent Claim
19 words
The method of claim 40 wherein no more than three of said dATP, dGTP, dCTP, and dTTP are used.
Claim 47
Dependent Claim
32 words
The method of claim 36 wherein said first nucleotide sequence is at least 10 nucleotides in length and the combined length of said first and second sequences is at least 20 nucleotides.
Claim 48
Dependent Claim
18 words
The method of claim 36 wherein said means for causing is an enzymatic means or a chemical means.
Claim 49
Dependent Claim
13 words
The method of claim 36 wherein said means for immobilizing is a surface.
Claim 50
Dependent Claim
28 words
The method of claim 36 wherein said reporter group is selected from the group consisting of fluorescers, chemiluminescers, catalysts, radioactive groups, enzymes, coenzymes substrates, and amplifiable nucleotide sequences.
Claim 51
Independent Claim
218 words
A method for detecting the presence of a target nucleotide sequence, which comprises: combining in a liquid medium (1) a sample suspected of containing a target nucleotide sequence, (2) a first nucleotide sequence complementary to a first portion of said target sequence, and (3) a second nucleotide sequence complementary to a portion of said target sequence other than and non-contiguous with said first portion, at least one of said first and said second nucleotide sequences having a small organic molecule bound thereto and the other being bound or having the capacity for binding to a reporter group, under conditions wherein said first and second nucleotide sequences bind to said target sequence, adding to said medium a polynucleotide polymerase and at least one nucleoside triphosphate under conditions wherein said second nucleotide sequence is extended to become contiguous with said first nucleotide sequence, adding to said medium a ligase under conditions wherein said first and second nucleotide sequences become covalently attached, adding to said medium a surface having bound thereto a binding partner for said small organic molecule under conditions wherein said first nucleotide sequence becomes bound to said surface, separating said surface from said medium, examining said surface for the presence of said reporter group, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
Claim 52
Independent Claim
218 words
A method for detecting the presence of a target nucleotide sequence, which comprises: combining in a liquid medium (1) a sample suspected of containing a target nucleotide sequence, (2) a first nucleotide sequence complementary to a first portion of said target sequence, and (3) a second nucleotide sequence complementary to a portion of said target sequence other than and non-contiguous with said first portion, at least one of said first and said second nucleotide sequences having a small organic molecule bound thereto and the other being bound or having the capacity for binding to a reporter group, under conditions wherein said first and second nucleotide sequences bind to said target sequence, adding to said medium a polynucleotide polymerase and at least one nucleoside triphosphate under conditions wherein said second nucleotide sequence is extended to become contiguous with said first nucleotide sequence, adding to said medium a ligase under conditions wherein said first and second nucleotide sequences become covalently attached, adding to said medium a surface having bound thereto a binding partner for said small organic molecule under conditions wherein said first nucleotide sequence becomes bound to said surface, separating said surface from said medium, examining said surface for the presence of said reporter group, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
Claim 53
Dependent Claim
14 words
The method of claim 46 wherein said target nucleotide sequence in DNA or RNA.
Claim 54
Dependent Claim
17 words
The method of claim 46 wherein said nucleoside triphosphate is selected from dATP, dGTP, dCTP and dTTP.
Claim 55
Dependent Claim
30 words
The method of claim 46 wherein said first nucleotide sequence is at least 10 and the combined length of said first and second nucleotide sequences is at least 20 nucleotides.
Claim 56
Dependent Claim
10 words
The method of claim 46 wherein said surface is bibulous.
Claim 57
Dependent Claim
17 words
The method of claim 46 wherein said small organic molecule has a molecular weight less than 1500.
Claim 58
Dependent Claim
22 words
The method of claim 46 wherein said small organic molecule is biotin and said binding partner bound to said surface is avidin.
Claim 59
Dependent Claim
28 words
The method of claim 46 wherein said reporter group is selected from the group consisting of fluorescers, chemiluminescers, catalysts, radioactive groups, enzymes, substrates, coenzymes, and amplifiable nucleotide sequences.
Claim 60
Dependent Claim
102 words
The method of claim 1 wherein said second polynucleotide contains or is a precursor to a polynucleotide containing a 3'-OH group and said method further comprises: (1) treating said covalently attached first and second nucleotide sequences with means to provide a 3'-OH group when said group is not present, a template nucleotide sequence complementary to said nucleotide sequence containing a 3'-OH group, a polynucleotide polymerase, nucleoside triphosphates, and means for cleaving a nucleotide sequence complementary to said template, under conditions for forming an extension of said nucleotide sequence containing a 3'-OH group and cleaving said extension, and (2) detecting said cleaved extension.
Claim 61
Dependent Claim
17 words
The method of claim 54 wherein said cleaved extension is about 6 to 30 nucleotides in length.
Claim 62
Dependent Claim
20 words
The method of claim 54 wherein said method is conducted under conditions for producing multiple copies of said cleaved extension.
Claim 63
Dependent Claim
21 words
The method of claim 2 wherein said first and second portions of said target nucleotide sequence are separated by one nucleotide.
Claim 64
Dependent Claim
181 words
The method of claim 1 wherein said second nucleotide sequence contains a detection sequence at a first end wherein said first end does not bind said target nucleotide sequence and is opposite a second end that binds to said target nucleotide sequence and said method further comprises: hybridizing said second nucleotide sequence with a third nucleotide sequence and a fourth nucleotide sequence, wherein said third nucleotide sequence contains a sequence complementary to said detection sequence and a different sequence and said fourth nucleotide sequence has a nucleotide sequence complementary to said different sequence and a nucleotide sequence identical to said detection sequence, with the proviso that one pair of complementary nucleotide sequences of the third and fourth nucleotide sequences is at the same end of each and a second pair of complementary nucleotide sequences of the third and fourth nucleotide sequences is at the other end of each, under conditions wherein said nucleotide sequences become ligated to each other and to said second nucleotide sequence when hybridized to said second nucleotide sequence and detecting said ligated third and/or fourth nucleotide sequences.
Claim 65
Independent Claim
152 words
A method for detecting a target nucleotide sequence in a polynucleotide, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence having first and second portions separated by one nucleotide, (b) a first polynucleotide having a sequence complementary to a first portion of said target nucleotide sequence, (c) a second polynucleotide having a sequence complementary to a second portion of said target nucleotide sequence other than and non-contiguous with said first portion, and (d) means for covalently attaching said first and second polynucleotides when said polynucleotides are hybridized with said target nucleotide sequence, under conditions wherein said first and second polynucleotides hybridize with said target nucleotide sequence and become covalently attached when said target nucleotide sequence is present; and determining the presence of covalently attached first and second polynucleotides, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
Claim 66
Independent Claim
152 words
A method for detecting a target nucleotide sequence in a polynucleotide, which comprises: providing in combination in a liquid medium (a) a sample suspected of containing a target nucleotide sequence having first and second portions separated by one nucleotide, (b) a first polynucleotide having a sequence complementary to a first portion of said target nucleotide sequence, (c) a second polynucleotide having a sequence complementary to a second portion of said target nucleotide sequence other than and non-contiguous with said first portion, and (d) means for covalently attaching said first and second polynucleotides when said polynucleotides are hybridized with said target nucleotide sequence, under conditions wherein said first and second polynucleotides hybridize with said target nucleotide sequence and become covalently attached when said target nucleotide sequence is present; and determining the presence of covalently attached first and second polynucleotides, the presence thereof indicating the presence of said target nucleotide sequence in said sample.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1996-02-09 | 1996-08-10 | 1997-02-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-02-09 | 2000-08-10 | 2001-02-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-02-09 | 2004-08-10 | 2005-02-09 | $7700.00 | Window Closed / Lapsed |