US6358679B1
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Methods for external controls for nucleic acid amplification
Assignee: Applied Biosystems LLC
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Filing: 2000-08-24
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Granted: 2002-03-19
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Expires: 2020-08-24
Abstract
Abstract Methods of nucleic acid amplification with external controls are provided that verify the absence or presence of specific target sequences, and correct primers and probes. A single-stranded, external control polynucleotide is amplified with primers of the same sequence as target primers. Probes with detectable labels and sequences specific for target and external control polynucleotides allow for detection and measurement. The primers and the detectable probe are adjacent or substantially adjacent when hybridized to the external control polynucleotide. Target and control amplicons may be detected by increased fluorescence induced by polymerase-mediated 5' nuclease cleavage or hybridization of a self-quenching probe complementary to both target and external control polynucleotides. A kit of PCR reagents can be dispensed into vessels for rapid and accurate nucleic acid amplification assay, with real-time or end-point measurements. The amplification control reagents, kits, and methods of the present invention provide positive and negative control tests which can be conducted concurrently with target amplification. Allelic differences at genetic loci can be detected, including single nucleotide polymorphisms (SNP).
Inventors
- 👤 Christian A. Heid
- 👤 Kenneth J. Livak
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 15:36
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/686; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 40
Claim 1
Independent Claim
160 words
A method for detecting a target polynucleotide sequence comprising the steps of: (a) amplifying a target polynucleotide with primer extension reagents in a first set of one or more vessels and amplifying an external control polynucleotide with the primer extension reagents in a second set of one or more vessels; wherein the primer extension reagents include a forward primer, a reverse primer, one or more detectable probes, a polymerase, and one or more nucleotide 5′-triphosphates; the forward primer and the detectable probe are separated by 0 to 5 nucleotides when hybridized to the external control polynucleotide, or its complement, and the reverse primer and the detectable probe are separated by 0 to 5 nucleotides when hybridized to the external control polynucleotide, or its complement; and the external control polynucleotide begins the amplification process as a single-stranded polynucleotide; and the external control polynucleotide is shorter than the target polynucleotide; and (b) detecting a signal from said one or more detectable probes.
Claim 2
Independent Claim
160 words
A method for detecting a target polynucleotide sequence comprising the steps of: (a) amplifying a target polynucleotide with primer extension reagents in a first set of one or more vessels and amplifying an external control polynucleotide with the primer extension reagents in a second set of one or more vessels; wherein the primer extension reagents include a forward primer, a reverse primer, one or more detectable probes, a polymerase, and one or more nucleotide 5′-triphosphates; the forward primer and the detectable probe are separated by 0 to 5 nucleotides when hybridized to the external control polynucleotide, or its complement, and the reverse primer and the detectable probe are separated by 0 to 5 nucleotides when hybridized to the external control polynucleotide, or its complement; and the external control polynucleotide begins the amplification process as a single-stranded polynucleotide; and the external control polynucleotide is shorter than the target polynucleotide; and (b) detecting a signal from said one or more detectable probes.
Claim 3
Dependent Claim
21 words
The method of claim 1 wherein said detectable probe is a self-quenching fluorescence probe comprising a reporter dye and a quencher.
Claim 4
Dependent Claim
23 words
The method of claim 2 wherein said polymerase cleaves said self-quenching fluorescence probes during amplification to separate said reporter dye from said quencher.
Claim 5
Dependent Claim
61 words
The method of claim 1 wherein the primer extension reagent of the second set of vessels comprises a first detectable probe and a second detectable probe; the sequence of the first probe differs from the second probe by one or more mismatches, insertions, or deletions, and the signal of the first probe is resolvable from the signal of the second probe.
Claim 6
Dependent Claim
66 words
The method of claim 2 wherein the primer extension reagent of the second set of vessels comprises a first self-quenching fluorescence probe and a second self-quenching fluorescence probe; the sequence of the first probe differs from the second probe by one or more mismatches, insertions, or deletions, and the reporter dye of the first probe is spectrally resolvable from the reporter dye of the second probe.
Claim 7
Dependent Claim
25 words
The method of claim 5 wherein the sequences of the first self-quenching fluorescence probe and second self-quenching fluorescence probe differ by a single nucleotide mismatch.
Claim 8
Dependent Claim
27 words
The method of claim 1 further comprising amplifying a second single-stranded external control polynucleotide with primer extension reagents in a third set of one or more vessels.
Claim 9
Dependent Claim
31 words
The method of claim 7 wherein the sequence of the first single-stranded external control polynucleotide differs from the second single-stranded external control polynucleotide by one or more mismatches, insertions, or deletions.
Claim 10
Dependent Claim
42 words
The method of claim 7 wherein the sequence portion of the first single-stranded external control polynucleotide complementary to a detectable probe differs by a single nucleotide from the sequence portion of the second single-stranded external control polynucleotide complementary to a detectable probe.
Claim 11
Dependent Claim
19 words
The method of claim 7 further comprising a fourth set of one or more vessels including primer extension reagents.
Claim 12
Dependent Claim
32 words
The method of claim 1 wherein the target polynucleotide is selected from the group consisting of a plasmid, a cDNA, a PCR product, genomic DNA, a restriction digest, and a ligation product.
Claim 13
Dependent Claim
30 words
The method of claim 1 wherein the forward primer or the reverse primer, and the detectable probe are adjacent when hybridized to the single-stranded external control polynucleotide, or its complement.
Claim 14
Dependent Claim
34 words
The method of claim 1 wherein the external control polynucleotide, or its complement, forms single-stranded overhangs consisting of 0 to about 10 nucleotides when hybridized to the forward primer or to the reverse primer.
Claim 15
Dependent Claim
20 words
The method of claim 1 wherein the forward primer and reverse primer are each 10 to 40 nucleotides in length.
Claim 16
Dependent Claim
17 words
The method of claim 2 wherein the self-quenching fluorescence probe is 10 to 40 nucleotides in length.
Claim 17
Dependent Claim
18 words
The method of claim 1 wherein the single-stranded external control polynucleotide is 30 to 110 nucleotides in length.
Claim 18
Dependent Claim
18 words
The method of claim 1 wherein the single-stranded external control polynucleotide is 50 to 70 nucleotides in length.
Claim 19
Dependent Claim
23 words
The method of claim 1 wherein a change in fluorescence intensity is detected as an indication of the presence of the target sequence.
Claim 20
Dependent Claim
27 words
The method of claim 1 wherein the products of the nucleic acid amplification of the target polynucleotide and the external control polynucleotide are detected by end-point analysis.
Claim 21
Dependent Claim
27 words
The method of claim 1 wherein the products of the nucleic acid amplification of the target polynucleotide and the external control polynucleotide are detected during real-time analysis.
Claim 22
Dependent Claim
26 words
The method of claim 1 wherein the products of the nucleic acid amplification of the target polynucleotide and the external control polynucleotide are detected by fluorescence.
Claim 23
Dependent Claim
18 words
The method of claim 1 wherein said nucleic acid polymerase is a thermostable polymerase with 5′ nuclease activity.
Claim 24
Dependent Claim
12 words
The method of claim 2 wherein said reporter is a xanthene dye.
Claim 25
Dependent Claim
13 words
The method of claim 23 wherein said xanthene dye is a fluorescein dye.
Claim 26
Dependent Claim
25 words
The method of claim 24 wherein said fluorescein dye is selected from the group consisting of: where X is an attached site to the probe.
Claim 27
Dependent Claim
24 words
The method of claim 2 wherein said quencher is selected from the group consisting of: where X is an attachment site to the probe.
Claim 28
Dependent Claim
19 words
The method of claim 2 wherein said reporter dye is separated from said quencher by at least 12 nucleotides.
Claim 29
Dependent Claim
23 words
The method of claim 2 wherein said reporter dye is attached at the 5′ terminus or 3′ terminus of the self-quenching fluorescence probe.
Claim 30
Dependent Claim
22 words
The method of claim 2 wherein said quencher is attached at the 5′ terminus or 3′ terminus of the self-quenching fluorescence probe.
Claim 31
Dependent Claim
10 words
The method of claim 2 wherein said quencher is non-fluorescent.
Claim 32
Dependent Claim
16 words
The method of claim 1 wherein the detectable probe is labelled with a minor groove binder.
Claim 33
Dependent Claim
17 words
The method of claim 2 wherein the self-quenching fluorescence probe is labelled with a minor groove binder.
Claim 34
Dependent Claim
22 words
The method of claim 32 wherein the self-quenching fluorescence probe is labelled with a minor groove binder at the 3′ tenninus nucleotide.
Claim 35
Dependent Claim
22 words
The method of claim 32 wherein the minor groove binder has the structure: where X is an attachment site to the probe.
Claim 36
Dependent Claim
13 words
The method of claim 1 where one or more nucleotide 5′-triphosphates are labelled.
Claim 37
Dependent Claim
20 words
The method of claim 35 wherein the label is a fluorescent dye, a quencher, biotin, or a minor groove binder.
Claim 38
Dependent Claim
14 words
The method of claim 1 wherein the vessels are located in a microwell tray.
Claim 39
Dependent Claim
18 words
The method of claim 1 wherein the primer extension reagents are delivered to the vessels by robotic means.
Claim 40
Dependent Claim
13 words
The method of claim 1 wherein amplification is conducted by a thermal cycler.
References Cited (Backward Citations)
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