US5972610A
Expired / Lapsed
US
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Use of nucleic acid analogues in the inhibition of nucleic acid amplification
Assignee: Roche Diagnostics GmbH
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Filing: 1997-10-08
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Granted: 1999-10-26
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Expires: 2017-10-08
Abstract
Abstract The present invention relates to the use of nucleic acid analogues in blocking nucleic acid amplification procedures and to diagnostic and analytical techniques based thereon. Also included are kits for use in the conduct of nucleic acid amplification reactions.
Inventors
- 👤 Ole Buchardt, deceased
- 👤 legal representative by Dorte Buchardt
- 👤 Michael Egholm
- 👤 Peter Eigil Nielsen
- 👤 Rolf Henrik Berg
- 👤 Christopher John Stanley
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 07:02
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6848; C07; C07H; C07H21/00; C12Q1/686
Claims Analysis
Total Claims: 33
Claim 1
Independent Claim
93 words
A method of inhibiting a nucleic acid amplification procedure, in which amplification procedure each strand of a double-stranded target nucleic acid has a region used as a first template for at least one primer which linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising hybridizing a nucleic acid analogue to a sequence of said first template or said at least one primer to inhibit linking or extension of the at least one primer during performance of the amplification procedure.
Claim 2
Independent Claim
93 words
A method of inhibiting a nucleic acid amplification procedure, in which amplification procedure each strand of a double-stranded target nucleic acid has a region used as a first template for at least one primer which linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising hybridizing a nucleic acid analogue to a sequence of said first template or said at least one primer to inhibit linking or extension of the at least one primer during performance of the amplification procedure.
Claim 3
Dependent Claim
60 words
The method of claim 1, wherein the hybrid formed between the nucleic acid analogue and the sequence of said first template or said at least one primer is more stable against heat denaturation than a hybrid between a conventional nucleic acid corresponding to the nucleic acid analogue and the sequence of said first template or said at least one primer.
Claim 4
Dependent Claim
16 words
The method of claim 1, wherein the amplification procedure is a PCR or an LCR procedure.
Claim 5
Dependent Claim
27 words
The method of claim 1, further comprising, before said hybridizing step, conducting said amplification procedure in the absence of said nucleic acid analogue to accumulate amplification product.
Claim 6
Dependent Claim
15 words
The method of claim 4, wherein said nucleic acid analogue hybridizes to said amplification product.
Claim 7
Independent Claim
141 words
A method of inhibiting amplification of a contaminating nucleic acid in an amplification procedure conducted using an amplification apparatus, in which amplification procedure each strand of a double-stranded nucleic acid has a region used as a first template for at least one primer which is linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising contacting a solution containing a nucleic acid analogue with said amplification apparatus to hybridize said nucleic acid analogue to any nucleic acid which may be present as a contamination to inhibit linking or extension of the contaminating nucleic acid during performance of the amplification procedure; thereafter washing the amplification apparatus; and thereafter conducting said amplification procedure using said amplification apparatus, wherein amplification of the contaminating nucleic acid is inhibited by the hybridizing step.
Claim 8
Independent Claim
141 words
A method of inhibiting amplification of a contaminating nucleic acid in an amplification procedure conducted using an amplification apparatus, in which amplification procedure each strand of a double-stranded nucleic acid has a region used as a first template for at least one primer which is linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising contacting a solution containing a nucleic acid analogue with said amplification apparatus to hybridize said nucleic acid analogue to any nucleic acid which may be present as a contamination to inhibit linking or extension of the contaminating nucleic acid during performance of the amplification procedure; thereafter washing the amplification apparatus; and thereafter conducting said amplification procedure using said amplification apparatus, wherein amplification of the contaminating nucleic acid is inhibited by the hybridizing step.
Claim 9
Dependent Claim
44 words
The method of claim 6, wherein the hybrid formed between the nucleic acid analogue and the contaminating nucleic acid is more stable against heat denaturation than a hybrid between a conventional nucleic acid corresponding to the nucleic acid analogue and the contaminating nucleic acid.
Claim 10
Independent Claim
96 words
A method of preventing a nucleic acid amplification product from serving as a template in a subsequent amplification procedure, in which subsequent amplification procedure each strand of a double-stranded nucleic acid has a region used as a first template for at least one primer which is linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising hybridizing a nucleic acid analogue to the nucleic acid amplification product to inhibit linking or extension of said amplification product during performance of the subsequent amplification procedure.
Claim 11
Independent Claim
96 words
A method of preventing a nucleic acid amplification product from serving as a template in a subsequent amplification procedure, in which subsequent amplification procedure each strand of a double-stranded nucleic acid has a region used as a first template for at least one primer which is linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising hybridizing a nucleic acid analogue to the nucleic acid amplification product to inhibit linking or extension of said amplification product during performance of the subsequent amplification procedure.
Claim 12
Dependent Claim
46 words
The method of claim 8, wherein the hybrid formed between the nucleic acid analogue and the nucleic acid amplification product is more stable against heat denaturation than a hybrid between a conventional nucleic acid corresponding to the nucleic acid analogue and the nucleic acid amplification product.
Claim 13
Independent Claim
132 words
A method of inhibiting amplification of a contaminating nucleic acid in a subsequent amplification procedure conducted in an environment, in which subsequent amplification procedure each strand of a double-stranded nucleic acid has a region used as a first template for at least one primer which is linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising contacting a nucleic acid analogue with the environment to hybridize said nucleic acid analogue to any nucleic acid which may be present as a contamination to inhibit linking or extension of the at least one primer during performance of the subsequent amplification procedure; and thereafter conducting the subsequent amplification procedure, wherein amplification of the contaminating nucleic acid is inhibited by the hybridizing step.
Claim 14
Independent Claim
132 words
A method of inhibiting amplification of a contaminating nucleic acid in a subsequent amplification procedure conducted in an environment, in which subsequent amplification procedure each strand of a double-stranded nucleic acid has a region used as a first template for at least one primer which is linked to another primer or extended with nucleotides to form a second template which is complementary to said first template, said method comprising contacting a nucleic acid analogue with the environment to hybridize said nucleic acid analogue to any nucleic acid which may be present as a contamination to inhibit linking or extension of the at least one primer during performance of the subsequent amplification procedure; and thereafter conducting the subsequent amplification procedure, wherein amplification of the contaminating nucleic acid is inhibited by the hybridizing step.
Claim 15
Dependent Claim
44 words
The method of claim 10, wherein the hybrid formed between the nucleic acid analogue and the contaminating nucleic acid is more stable against heat denaturation than a hybrid between a conventional nucleic acid corresponding to the nucleic acid analogue and the contaminating nucleic acid.
Claim 16
Independent Claim
166 words
A method of detecting the presence or absence of a target nucleic acid sequence, comprising (a) conducting an amplification procedure using (1) a double-stranded nucleic acid comprising a nucleic acid region, which nucleic acid region may contain the target nucleic acid sequence, (2) at least one primer, and (3) a nucleic acid analogue comprising a sequence which is complementary to said target nucleic acid sequence, wherein when the target nucleic acid sequence is present, then the nucleic acid analogue hybridizes with the target nucleic acid sequence to inhibit the at least one primer from linking to another primer or extending with nucleotides during performance of the amplification procedure, or when the target nucleic acid sequence is absent, then the at least one primer hybridizes with the nucleic acid region and is linked to another primer or extended with nucleotides to form an amplification product; and (b) determining the presence or absence of amplification product to detect the presence or absence of the target nucleic acid sequence.
Claim 17
Independent Claim
166 words
A method of detecting the presence or absence of a target nucleic acid sequence, comprising (a) conducting an amplification procedure using (1) a double-stranded nucleic acid comprising a nucleic acid region, which nucleic acid region may contain the target nucleic acid sequence, (2) at least one primer, and (3) a nucleic acid analogue comprising a sequence which is complementary to said target nucleic acid sequence, wherein when the target nucleic acid sequence is present, then the nucleic acid analogue hybridizes with the target nucleic acid sequence to inhibit the at least one primer from linking to another primer or extending with nucleotides during performance of the amplification procedure, or when the target nucleic acid sequence is absent, then the at least one primer hybridizes with the nucleic acid region and is linked to another primer or extended with nucleotides to form an amplification product; and (b) determining the presence or absence of amplification product to detect the presence or absence of the target nucleic acid sequence.
Claim 18
Dependent Claim
46 words
The method of claim 12, wherein the hybrid formed between the nucleic acid analogue and the target nucleic acid sequence is more stable against heat denaturation than a hybrid between a conventional nucleic acid corresponding to the nucleic acid analogue and the target nucleic acid sequence.
Claim 19
Dependent Claim
102 words
The method of claim 12, wherein the target nucleic acid sequence, if present, is between a pair of primer binding sites, and the method comprises sequentially detecting the presence or absence of each of a plurality of sequences within the target nucleic acid sequence by conducting a series of amplification procedures using the same primers and different nucleic acid analogues for each of the series, each of which is complementary to a different one of the plurality of sequences, and determining whether amplification of each of the plurality of sequences has occurred to detect the presence or absence of the each sequence.
Claim 20
Dependent Claim
57 words
The method of claim 12, wherein the conducting step (a) further comprises including a control nucleic acid which undergoes amplification using the same at least one primer, to produce a second amplification product which is distinguishable from the amplification product comprising the nucleic acid region, and the determining step (b) further comprises determining the second amplification product.
Claim 21
Independent Claim
108 words
A method of conducting an asymmetric nucleic acid amplification procedure, comprising providing a double-stranded nucleic acid, wherein each strand of the double-stranded nucleic acid has a region used as a first template for a first primer which is extended with nucleotides to form a second template which is complementary to said first template; amplifying the double-stranded nucleic acid for a number of amplification cycles to produce amplification product; combining the amplification product with a nucleic acid analogue which hybridizes to the first primer to inhibit extension of the first primer with nucleotides during performance of further amplification cycles, and continuing the amplification procedure for the further amplification cycles.
Claim 22
Independent Claim
108 words
A method of conducting an asymmetric nucleic acid amplification procedure, comprising providing a double-stranded nucleic acid, wherein each strand of the double-stranded nucleic acid has a region used as a first template for a first primer which is extended with nucleotides to form a second template which is complementary to said first template; amplifying the double-stranded nucleic acid for a number of amplification cycles to produce amplification product; combining the amplification product with a nucleic acid analogue which hybridizes to the first primer to inhibit extension of the first primer with nucleotides during performance of further amplification cycles, and continuing the amplification procedure for the further amplification cycles.
Claim 23
Dependent Claim
42 words
The method of claim 16, wherein the hybrid formed between the nucleic acid analogue and the first primer is more stable against heat denaturation than a hybrid between a conventional nucleic acid corresponding to the nucleic acid analogue and the first primer.
Claim 24
Independent Claim
69 words
A method of inhibiting a polymerase-mediated extension procedure of a primer which is hybridized tc a nucleic acid template, comprising providing an extension procedure reaction mixture comprising a nucleic acid template, a primer and a polymerase; and combining with the mixture a nucleic acid analogue which hybridizes to the primer or the nucleic acid template to inhibit extension of the primer with nucleotides during performance of the extension procedure.
Claim 25
Independent Claim
69 words
A method of inhibiting a polymerase-mediated extension procedure of a primer which is hybridized tc a nucleic acid template, comprising providing an extension procedure reaction mixture comprising a nucleic acid template, a primer and a polymerase; and combining with the mixture a nucleic acid analogue which hybridizes to the primer or the nucleic acid template to inhibit extension of the primer with nucleotides during performance of the extension procedure.
Claim 26
Dependent Claim
50 words
The method of claim 18, wherein the hybrid formed between the nucleic acid analogue and the primer or the nucleic acid template is more stable against heat denaturation than a hybrid between a conventional nucleic acid corresponding to the nucleic acid analogue and the primer or the nucleic acid template.
Claim 27
Dependent Claim
15 words
The method of claim 1, wherein the nucleic acid analogue comprises a peptide nucleic acid.
Claim 28
Dependent Claim
15 words
The method of claim 6, wherein the nucleic acid analogue comprises a peptide nucleic acid.
Claim 29
Dependent Claim
15 words
The method of claim 8, wherein the nucleic acid analogue comprises a peptide nucleic acid.
Claim 30
Dependent Claim
15 words
The method of claim 10, wherein the nucleic acid analogue comprises a peptide nucleic acid.
Claim 31
Dependent Claim
15 words
The method of claim 12, wherein the nucleic acid analogue comprises a peptide nucleic acid.
Claim 32
Dependent Claim
15 words
The method of claim 16, wherein the nucleic acid analogue comprises a peptide nucleic acid.
Claim 33
Dependent Claim
15 words
The method of claim 18, wherein the nucleic acid analogue comprises a peptide nucleic acid.
References Cited (Backward Citations)
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| 3.5 Years (Year 4) | 2002-10-25 | 2003-04-26 | 2003-10-26 | $2000.00 | Window Closed / Lapsed |
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