US5508178A
Expired / Lapsed
US
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Nucleic acid amplification using single primer
Assignee: Siemens Healthcare Diagnostics GmbH Germany
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Filing: 1994-02-09
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Granted: 1996-04-16
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Expires: 2014-02-09
Abstract
Abstract A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which has at least a 10 base sequence hybridizable with a second sequence flanking the 3'-end of the single stranded polynucleotide, the second sequence being partially or fully complementary with at least a 10 base first sequence flanking the 5' end of the single stranded polynucleotide, (b) dissociating the extended polynucleotide primer and the single stranded polynucleotide, (c) repeating step a and (d) dissociating the extended polynucleotide primer and the copy of the single stranded polynucleotide.
Inventors
- 👤 Samuel Rose
- 👤 Thomas C. Goodman
- 👤 Linda M. Western
- 👤 Martin Becker
- 👤 Edwin F. Ullman
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 11:42
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6858; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
144 words
A method of producing at least one copy of a single stranded polynucleotide which comprises: (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along a single stranded polynucleotide, flanked at the 5'-end and the 3'-end, respectively, by a first flanking sequence and a second flanking sequence, an extension of a polynucleotide primer at least the 3'-end of said polynucleotide primer having at least a 10 base sequence hybridizable with a 10 base sequence of said second flanking sequence, said 10 base sequence of said second flanking sequence being at least partially complementary, and thus hybridizable, with said first flanking sequence; (b) dissociating said extended polynucleotide primer and said single stranded polynucleotide; and (c) repeating step a wherein said polynucleotide primer hybridizes to and is extended along (1) said single stranded polynucleotide and (2) said extension of said polynucleotide primer.
Claim 2
Independent Claim
144 words
A method of producing at least one copy of a single stranded polynucleotide which comprises: (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along a single stranded polynucleotide, flanked at the 5'-end and the 3'-end, respectively, by a first flanking sequence and a second flanking sequence, an extension of a polynucleotide primer at least the 3'-end of said polynucleotide primer having at least a 10 base sequence hybridizable with a 10 base sequence of said second flanking sequence, said 10 base sequence of said second flanking sequence being at least partially complementary, and thus hybridizable, with said first flanking sequence; (b) dissociating said extended polynucleotide primer and said single stranded polynucleotide; and (c) repeating step a wherein said polynucleotide primer hybridizes to and is extended along (1) said single stranded polynucleotide and (2) said extension of said polynucleotide primer.
Claim 3
Dependent Claim
24 words
The method of claim 1 wherein the last 10 nucleotides of the 3'-end of said polynucleotide primer are complementary to said second flanking sequence.
Claim 4
Dependent Claim
18 words
The method of claim 1 wherein said first flanking sequence is fully complementary to said second flanking sequence.
Claim 5
Dependent Claim
19 words
The method of claim 1 which further comprises the step of dissociating said extended polynucleotide primer and said copy.
Claim 6
Dependent Claim
15 words
The method of claim 1 wherein said single stranded polynucleotide or said copy is DNA.
Claim 7
Dependent Claim
17 words
The method of claim 5 wherein the product formed after repeating step (a) contains an inverted repeat.
Claim 8
Dependent Claim
20 words
The method of claim 1 wherein said hybridizable sequence of said polynucleotide primer is 15 to 100 nucleotides in length.
Claim 9
Dependent Claim
24 words
The method of claim 1 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.
Claim 10
Dependent Claim
29 words
The method of claim 1 wherein said method is carried out at a substantially excess concentration of said polynucleotide primer relative to the concentration of said single stranded polynucleotide.
Claim 11
Dependent Claim
31 words
A method comprising repetition of the steps of claim 1 wherein the number of said copies of said single stranded polynucleotide is increased by at least a factor of a thousand.
Claim 12
Independent Claim
243 words
A method of producing multiple copies of a polynucleotide sequence, which comprises: (a) providing in combination (1) a single stranded polynucleotide having said polynucleotide sequence and being flanked at each end by at least partially complementary first and second flanking sequences, (2) a polynucleotide primer at least a 10 base portion of which at its 3'-end is hybridizable to a 10 base sequence of that member of said first and second flanking sequences that is at the 3'-end of said single stranded polynucleotide, said 10 base sequence being hybridizable to a 10 base sequence of that member of said first and second flanking sequences that is at the 5'-end of said single stranded polynucleotide, (3) nucleoside triphosphates, and (4) template dependent polynucleotide polymerase and (b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said single stranded polynucleotide from any complementary sequences, (2) hybridizing said polynucleotide primer with the flanking sequence at the 3' end of said single stranded polynucleotide, (3) extending said polynucleotide primer along said single stranded polynucleotide to provide a first extended polynucleotide primer, (4) dissociating said first extended polynucleotide primer and said single stranded polynucleotide, (5) hybridizing said first extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said first extended polynucleotide primer to provide a second extended polynucleotide primer, (7) dissociating said second extended polynucleotide primer from said first extended polynucleotide primer, and (8) repeating steps (5)-(7) above.
Claim 13
Independent Claim
243 words
A method of producing multiple copies of a polynucleotide sequence, which comprises: (a) providing in combination (1) a single stranded polynucleotide having said polynucleotide sequence and being flanked at each end by at least partially complementary first and second flanking sequences, (2) a polynucleotide primer at least a 10 base portion of which at its 3'-end is hybridizable to a 10 base sequence of that member of said first and second flanking sequences that is at the 3'-end of said single stranded polynucleotide, said 10 base sequence being hybridizable to a 10 base sequence of that member of said first and second flanking sequences that is at the 5'-end of said single stranded polynucleotide, (3) nucleoside triphosphates, and (4) template dependent polynucleotide polymerase and (b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said single stranded polynucleotide from any complementary sequences, (2) hybridizing said polynucleotide primer with the flanking sequence at the 3' end of said single stranded polynucleotide, (3) extending said polynucleotide primer along said single stranded polynucleotide to provide a first extended polynucleotide primer, (4) dissociating said first extended polynucleotide primer and said single stranded polynucleotide, (5) hybridizing said first extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said first extended polynucleotide primer to provide a second extended polynucleotide primer, (7) dissociating said second extended polynucleotide primer from said first extended polynucleotide primer, and (8) repeating steps (5)-(7) above.
Claim 14
Dependent Claim
14 words
The method of claim 11 wherein said first and second sequences are fully complementary.
Claim 15
Dependent Claim
21 words
The method of claim 11 wherein said single stranded polynucleotide sequence or said first or second extended polynucleotide primer is DNA.
Claim 16
Dependent Claim
23 words
The method of claim 11 wherein said first extended polynucleotide primer when hybridized with said second extended polynucleotide primer contains an inverted repeat.
Claim 17
Dependent Claim
16 words
The method of claim 11 wherein said polynucleotide primer is 15 to 100 nucleotides in length.
Claim 18
Dependent Claim
24 words
The method of claim 11 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.
Claim 19
Dependent Claim
29 words
The method of claim 11 wherein said method is carried out at a substantially excess concentration of said polynucleotide primer relative to the concentration of said single stranded polynucleotide.
Claim 20
Dependent Claim
25 words
The method of claim 11 wherein the number of said copies of said polynucleotide sequence is increased by at least a factor of a thousand.
Claim 21
Independent Claim
240 words
A method of producing multiple copies of a polynucleotide sequence in a polynucleotide, said sequence being flanked at each end by a different member of a pair of flanking sequences that are at least partially complementary to each other, which comprises: (a) combining said polynucleotide with a single polynucleotide primer having at least a terminal sequence at its 3'end at least partially complementary to and hybridizable with at least a portion of the member of said pair of flanking sequences at the 3'-end of said polynucleotide sequence, said portion being hybridizable with the member of said pair of flanking sequences at the 5'-end of said polynucleotide sequence, nucleoside triphosphates, and template dependent polynucleotide polymerase, (b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said polynucleotide sequence from any sequence with which it is hybridized to provide a single stranded polynucleotide, (2) hybridizing said polynucleotide primer with the flanking sequence at the 3'-end of said single stranded polynuycleotide, (3) extending said polynucleotide primer along said single stranded polynucleotide to provide an extended polynucleotide primer, (4) dissociating said first extended polynucleotide primer and said single stranded polynucleotide, (5) hybridizing said first extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said first extended polynucleotide primer to provide a second extended polynucleotide primer, (7) dissociating said second extended polynucleotide primer from said first extended polynuycleotide primer, and (8) repeating steps (5)-(7) above.
Claim 22
Independent Claim
240 words
A method of producing multiple copies of a polynucleotide sequence in a polynucleotide, said sequence being flanked at each end by a different member of a pair of flanking sequences that are at least partially complementary to each other, which comprises: (a) combining said polynucleotide with a single polynucleotide primer having at least a terminal sequence at its 3'end at least partially complementary to and hybridizable with at least a portion of the member of said pair of flanking sequences at the 3'-end of said polynucleotide sequence, said portion being hybridizable with the member of said pair of flanking sequences at the 5'-end of said polynucleotide sequence, nucleoside triphosphates, and template dependent polynucleotide polymerase, (b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said polynucleotide sequence from any sequence with which it is hybridized to provide a single stranded polynucleotide, (2) hybridizing said polynucleotide primer with the flanking sequence at the 3'-end of said single stranded polynuycleotide, (3) extending said polynucleotide primer along said single stranded polynucleotide to provide an extended polynucleotide primer, (4) dissociating said first extended polynucleotide primer and said single stranded polynucleotide, (5) hybridizing said first extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said first extended polynucleotide primer to provide a second extended polynucleotide primer, (7) dissociating said second extended polynucleotide primer from said first extended polynuycleotide primer, and (8) repeating steps (5)-(7) above.
Claim 23
Dependent Claim
10 words
The method of claim 19 wherein said polynucleotide is DNA.
Claim 24
Dependent Claim
16 words
The method of claim 19 wherein said polynucleotide primer is 15 to 100 nucleotides in length.
Claim 25
Dependent Claim
24 words
The method of claim 19 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.
Claim 26
Dependent Claim
27 words
The method of claim 19 wherein said method is carried out at a substantially excess concentration of said polynucleotide primer relative to the concentration of said polynucleotide.
Claim 27
Dependent Claim
30 words
The method of claim 19 wherein the concentration of reagents and at least one temperature are chosen to substantially optimize binding of said polynucleotide primer to said single stranded polynucleotide.
Claim 28
Dependent Claim
19 words
The method of claim 19 which further comprises the step of detecting at least one of said extended primers.
References Cited (Backward Citations)
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