EP0382433A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Detection of nucleic acid sequences using fluorescence polarisation
Assignee: Syngenta Ltd
•
Filing: 1990-02-02
•
Granted: —
•
Expires: 2010-02-02
Abstract
Abstract Assay method for the detection of nucleic acid sequences in homogeneous solution. The method comprises the use of fluorescence polarisation to detect hybridisation of a fluorescent nucleic acid probe or to detect fluorescent primer extension products. Assay kits for use in the above methods.
Inventors
- 👤 Andrew John Garman
- 👤 Robert Stanley Moore
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 09:51
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6827; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
51 words
A method for the detection of a target nucleic acid sequence in a sample which method comprises contacting the sample nucleic acid with a fluorescent nucleotide probe capable of hybridisation with the target nucleic acid sequence in homogeneous solution and detecting the presence or absence of such hybridisation by fluorescence polarisation.
Claim 2
Independent Claim
51 words
A method for the detection of a target nucleic acid sequence in a sample which method comprises contacting the sample nucleic acid with a fluorescent nucleotide probe capable of hybridisation with the target nucleic acid sequence in homogeneous solution and detecting the presence or absence of such hybridisation by fluorescence polarisation.
Claim 3
Dependent Claim
29 words
A method as claimed in claim 1 which comprises the use of two or more fluorescently labelled probes which hybridise to adjacent regions of the target nucleic acid sequence.
Claim 4
Dependent Claim
41 words
A method as claimed in claim 1 or claim 2 wherein one or more blocking polynucleotides are hybridised to the sample target nucleic acid strand to prevent annealing of the sample nucleic acid strand complementary to the target nucleic acid sequence.
Claim 5
Independent Claim
73 words
A method for the detection of a target nucleic acid sequence in a sample, which method comprises contacting the sample with (i) a complementary polynucleotide capable of hybridisation to the target nucleotide sequence and (ii) a fluorescent polynucleotide probe capable of hybridisation to the same region on the complementary polynucleotide, in homogeneous solution and detecting by fluorescence polarisation the presence or absence of hybridisation of the fluorescent polynucleotide probe with the complementary polynucleotide.
Claim 6
Independent Claim
73 words
A method for the detection of a target nucleic acid sequence in a sample, which method comprises contacting the sample with (i) a complementary polynucleotide capable of hybridisation to the target nucleotide sequence and (ii) a fluorescent polynucleotide probe capable of hybridisation to the same region on the complementary polynucleotide, in homogeneous solution and detecting by fluorescence polarisation the presence or absence of hybridisation of the fluorescent polynucleotide probe with the complementary polynucleotide.
Claim 7
Independent Claim
25 words
A method as claimed in any one of the previous claims wherein the target nucleic acid sequence is a product of sample nucleic acid amplification.
Claim 8
Dependent Claim
24 words
A method as claimed in claim 5 wherein the target nucleic acid sequence derived from sample nucleic acid amplification is essentially single stranded DNA.
Claim 9
Independent Claim
36 words
A method as claimed in any one of claims 1-3, 5 and 6 wherein the hybridised species to be detected by fluorescence polarisation includes one or more macromolecule-polynucleotide conjugates hybridised to the target nucleic acid sequence.
Claim 10
Independent Claim
21 words
A method as claimed in any one of claims 4-6 wherein one or more macromolecules are attached to the complementary polynucleotide.
Claim 11
Dependent Claim
16 words
A method as claimed in claim 7 or claim 8 wherein the macromolecule(s) are covalently attached.
Claim 12
Independent Claim
34 words
A method as claimed in any one of claims 5 and 6 when dependent on any one of claims 1-3 and wherein one or more macromolecules are attached to the target nucleic acid sequence.
Claim 13
Dependent Claim
35 words
A method as claimed in claim 10 wherein a specific binding substance is incorporated into the target nucleic acid sequence before or during amplification and contacted with a complementary specific binding substance-macromolecule conjugate after amplification.
Claim 14
Independent Claim
52 words
A method for the detection of a target nucleic acid sequence in a sample which comprises contacting sample nucleic acid with a fluorescent polynucleotide primer capable of hybridisation with the target sequence, subjecting any hybrid formed to primer extension and detecting the presence or absence of primer extended product by fluorescence polarisation.
Claim 15
Independent Claim
52 words
A method for the detection of a target nucleic acid sequence in a sample which comprises contacting sample nucleic acid with a fluorescent polynucleotide primer capable of hybridisation with the target sequence, subjecting any hybrid formed to primer extension and detecting the presence or absence of primer extended product by fluorescence polarisation.
Claim 16
Dependent Claim
31 words
A method as claimed in claim 12 which comprises a polymerase chain reaction and wherein a first fluorescent polynucleotide primer is employed at a lower concentration than a second polynucleotide primer.
Claim 17
Dependent Claim
18 words
A method as claimed in claim 13 wherein the second polynucleotide primer is present in 10-100 fold excess.
Claim 18
Independent Claim
52 words
A method as claimed in any one of claims 12-14 wherein two polynucleotide primers are employed each priming opposite strands of the sample nucleic acid and both primers are incorporated into the amplified product and wherein one primer is labelled with a fluorophore and a macromolecule is attached to the other primer.
Claim 19
Dependent Claim
22 words
A method as claimed in claim 15 wherein the macromolecule is non-covalently attached via specific binding substances to the primer after amplification.
Claim 20
Dependent Claim
23 words
A method as claimed in claim 11 or claim 16 wherein the specific binding substances are (i) biotin and (ii) avidin or streptavidin.
Claim 21
Independent Claim
18 words
A method as claimed in any one of the previous claims wherein the sample is a biological sample.
Claim 22
Independent Claim
32 words
A method as claimed in any one of the previous claims wherein the target nucleic acid sequence is an allele of a genetic locus indicative of an inherited disease, disorder or predisposition.
Claim 23
Independent Claim
23 words
A method as claimed in any one of the previous claims and performed as a quantitative assay of the target nucleic acid sequence.
Claim 24
Independent Claim
26 words
A method as claimed in any one of the previous claims wherein more than one target nucleic acid sequence is assayed using polynucleotides with different fluorophores.
Claim 25
Independent Claim
18 words
A method as claimed in any one of the previous claims wherein the fluorophore is fluorescein or rhodamine.
Claim 26
Dependent Claim
53 words
An assay kit which comprises an appropriately buffered solution of probe(s) and/or primer(s) and/or complementary polynucleotides, other optional reagents, other optional solutions for the pre-treatment of the sample nucleic acid, other optional buffer solutions together with appropriate packaging and instructions for performing a method as claimed in any one of the preceding claims.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.