EP0469755A1
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Method producing a polynucleotide for use in single primer amplification
Assignee: Siemens Healthcare Diagnostics GmbH Germany
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Filing: 1991-07-18
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Granted: —
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Expires: 2011-07-18
Abstract
Abstract A method is disclosed for producing a single stranded polydeoxynucleotide having two segments that are non-contiguous and complementary with each other. The method comprises the steps of providing in combination (1) a polynucleotide having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5′ of S1 and is at least ten deoxynucleotides long and (2) an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3′-end of the extender probe is hybridizable with S1 and the other of the deoxynucleotide sequences is homologous to S2 and (b) extending the extender probe along the polynucleotide. The method can also comprise providing in the combination a polydeoxynucleotide primer capable of hybridizing at least at its 3′-end with a nucleotide sequence complementary to S2 under conditions where (1) the extended extender probe is rendered single stranded, (2) the polydeoxynucleotide primer hybridizes with and is extended along the extended extender probe to form a duplex comprising extended primer, (3) the extended primer is dissociated from the duplex, and (4) the primer hybridizes with and is extended along the extended primer to form a duplex comprising extended primer, and repeating steps (3) and (4). The method finds particular application in the detection of polynucleotide analytes.
Inventors
- 👤 Samuel Rose
- 👤 Linda M. Western
- 👤 Martin Becker
- 👤 Edwin F. Ullman
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-04 19:16
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6853; C12N; C12N15/00; C12N15/09; C12N15/10; C12Q1/686
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
95 words
A method for producing a single stranded polydeoxynucleotide having two segments that are non-contiguous and complementary with each other, said method comprising the steps of: providing in combination a polynucleotide having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5′ of S1 and is at least ten nucleotides long and an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3′ end of said extender probe is hybridizable with S1 and the other of said deoxynucleotide sequences is homologous to S2 and extending said extender probe along said polynucleotide.
Claim 2
Dependent Claim
92 words
The method of Claim 1 which further comprises providing in said combination a polydeoxynucleotide primer capable of hybridizing at least at its 3′-end with a nucleotide sequence complementary to S2 under conditions where (1) said extended extender probe is rendered single stranded, (2) said polydeoxynucleotide primer hybridizes with and is extended along said extended extender probe to form a duplex comprising extended primer, (3) said extended primer is dissociated from said duplex, and (4) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer.
Claim 3
Dependent Claim
14 words
The method of Claim 1 or 2 wherein steps (3) and (4) are repeated.
Claim 4
Independent Claim
25 words
The method of any one of the preceding claims wherein the concentration of said extender probe is substantially lower than that of said polydeoxynucleotide primer.
Claim 5
Independent Claim
26 words
The method of any one of the preceding claims wherein the concentration of said extender probe is less than one percent that of said polydeoxynucleotide primer.
Claim 6
Dependent Claim
215 words
A method according to Claim 1 for producing multiple copies of a single stranded polydeoxynucleotide having two segments that are non-contiguous and complementary with each other, said method comprising the step of: providing in combination, either concomitantly or wholly or partially sequentially, a polynucleotide having two non-contiguous, non-complementary nucleotide sequences, S1 and S2, wherein S2 is 5′ of S1 and is at least ten nucleotides long, an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3′ end of said extender probe is hybridizable with S1 and the other of said nucleotide sequences is homologous to S2 and not complementary to said polynucleotide, a polydeoxynucleotide primer capable of hybridizing at least at its 3′ end with a nucleotide sequence complementary to S2, DNA polymerase, and deoxynucleoside triphosphates under conditions where (a) said extender probe is extended along said polynucleotide to form a duplex, (b) said extended extender probe is dissociated from said duplex, (c) said polydeoxynucleotide primer hybridizes with and is extended along said extended extender probe to form a second duplex comprising extended primer (d) said extended primer is dissociated from said second duplex, and (e) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer, and steps (d) and (e) are repeated.
Claim 7
Dependent Claim
16 words
The method of Claim 6 wherein steps (d) and (e) are repeated at least three times.
Claim 8
Dependent Claim
21 words
The method of Claim 6 or 7 wherein at least a fifteen deoxynucleotide sequence of said extender probe hybridizes with S1.
Claim 9
Dependent Claim
28 words
The method of Claim 6, 7 or 8 wherein said polydeoxynucleotide primer contains at least a fifteen deoxynucleotide sequence capable of hybridizing with a sequence complementary to S2.
Claim 10
Independent Claim
14 words
The method of any one of the Claims 6-9 wherein said polynucleotide is DNA.
Claim 11
Dependent Claim
220 words
A method according to Claim 1 for detecting the presence of a target nucleotide sequence in a medium suspected of containing said target nucleotide sequence, said target nucleotide sequence having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5′ of S1 and at least 10 nucleotides long, said method comprising the steps of: (a) providing in combination, either concomitantly or wholly or partially sequentially, said medium, an extender probe having two deoxynucleotide sequences wherein the sequence at the 3′ end of said extender probe is hybridizable with S1 and the other of said deoxynucleotide sequences is homologous to S2 and not complementary to said target nucleotide sequence, a polydeoxynucleotide primer capable of hybridizing with a nucleotide sequence complementary to S2, DNA polymerase and deoxynucleoside triphosphates under conditions wherein (1) said extender probe is extended along said polynucleotide to form a duplex, (2) said extended extender probe is dissociated from said duplex, (3) said primer hybridizes with and is extended along said extended extender probe to form a duplex comprising extended primer, (4) said extended primer is dissociated from said duplex, and (5) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer and steps (4) and (5) are repeated, and (b) examining for the presence of said extended primer.
Claim 12
Dependent Claim
17 words
The method of Claim 11 wherein S1 and S2 each respectively contain from 10 to 100 nucleotides.
Claim 13
Dependent Claim
29 words
The method of Claim 11 or 12 wherein the concentration of said extender probe is less than 1nM and the concentration of said polydeoxynucleotide primer is greater than 100nM.
Claim 14
Independent Claim
19 words
The method of any one of the Claims 11-13 wherein said polydeoxynucleotide primer is labeled with a reporter molecule.
Claim 15
Independent Claim
29 words
The method of any one of the Claims 11-14 wherein said polydeoxynucleotide primer contains a nucleotide sequence other than the sequence that hybridizes with said sequence complementary to S2.
Claim 16
Dependent Claim
42 words
The method of Claim 15 wherein said presence of said extended primer is detected by examining for a reporter molecule covalently bonded to a nucleotide sequence that is complementary to a portion of said target nucleotide sequence other than S1 or S2.
Claim 17
Dependent Claim
253 words
A method according to Claim 1 for detecting the presence of a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte, said method comprising the steps of: (a) treating a medium containing said sample to form a single stranded target nucleotide sequence from said polynucleotide analyte, if present, said target nucleotide sequence having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5′ of S1, and is at least ten nucleotides long, (b) combining said medium with an extender probe having two deoxynucleotide sequences wherein the sequence at the 3′ end of said extender probe is hybridizable with S1 and the other of said deoxynucleotide sequence is homologous to S2 and not complementary to said target sequence, a polydeoxynucleotide primer capable of hybridizing with a nucleotide sequence complementary to S2, deoxynucleoside triphosphates, and DNA template dependent polydeoxynucleotide polymerase under conditions wherein (1) said extender probe is hybridized with and is extended along said target nucleotide sequence to form a duplex, (2) said extended extender probe is dissociated from said duplex, (3) said primer hybridizes with and is extended along said extended extender probe to form a duplex comprising extended primer, (4) said extended primer is dissociated from said duplex, and (5) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer and steps (4) and (5) are repeated, wherein steps (a) and (b) are performed concomitantly or wholly or partially sequentially, and (c) examining for the presence of said extended primer.
Claim 18
Dependent Claim
16 words
The method of Claim 17 wherein steps (4) and (5) are repeated less than 30 times.
Claim 19
Dependent Claim
19 words
The method of Claim 17 or 18 wherein said polynucleotide analyte is RNA and said medium includes reverse transcriptase.
Claim 20
Independent Claim
19 words
The method of any one of the Claims 17-19 wherein said deoxynucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.
Claim 21
Independent Claim
33 words
The method of any one of the Claims 17-20 wherein steps (4) and 5) are repeated such that the number of said duplexes formed is increased by at least a factor of 1000.
Claim 22
Dependent Claim
35 words
The method of Claim 14 wherein said reporter molecule is selected from the group consisting of fluorescers, chemiluminescers, promoters, co-enzymes, radioactive substances, amplifiable polynucleotide sequences, small organic molecules, catalysts and polynucleotide sequences coding for catalysts.
Claim 23
Independent Claim
18 words
The method of any one of the Claims 17-22 wherein said polydeoxynucleotide primer is labeled with a ligand.
Claim 24
Independent Claim
29 words
The method of any one of the Claims 17-23 wherein said polydeoxynucleotide primer contains a nucleotide sequence other than the sequence that hybridizes with said sequence complementary to S2.
Claim 25
Dependent Claim
30 words
The method of Claim 24 wherein said nucleotide sequence of said polydeoxynucleotide primer contains a sequence that, when hybridized to its complementary sequence, can be bound specifically by a receptor.
Claim 26
Dependent Claim
19 words
The method of Claim 25 wherein said receptor is selected from the group consisting of repressors, activators, and nucleases.
Claim 27
Dependent Claim
44 words
The method of Claim 24 wherein said nucleotide sequence of said polydeoxynucleotide primer contains a sequence that when hybridized to its complementary sequence, can be bound specifically by a receptor, and said extended primer is detected by binding said receptor to said extended primer.
Claim 28
Dependent Claim
76 words
A kit comprising in packaged combination: an extender probe having at its 3′ end a sequence hybridizable with a first sequence in a target nucleotide sequence and having a sequence that is homologous to a second sequence of said target nucleotide sequence, wherein in said target nucleotide sequence said second sequence is 5′ and non-contiguous with said first sequence and a polydeoxynucleotide primer capable of hybridizing with a sequence that is complementary with said second sequence.
Claim 29
Dependent Claim
11 words
The kit of Claim 28 which comprises template dependent DNA polymerase.
Claim 30
Dependent Claim
11 words
The kit of Claim 28 or 29 which comprises deoxynucleoside triphosphates.
References Cited (Backward Citations)
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