EP0124221A1
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Method of detecting a polynucleotide sequence and labelled polynucleotides useful therein
Assignee: Blair Malcolm A D
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Filing: 1984-03-09
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Granted: —
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Expires: 2004-03-09
Abstract
Abstract A method of detecting nucleic acid sequences is disclosed and enzyme-labelled reagents for use therein. According to the invention sample polynucleotide is first hybridised with a polynucleotide probe comprising a nucleic acid sequence complementary to that which is to be detected, and having a polynucleotide tail, preferably a single stranded poly(dA) or poiy(dT) tail. Following attachment of the probe to the sample, an enzyme-containing marker, is attached to the probe, the marker comprising a polynucleotide having a single-stranded portion, to the base groups of which is or are attached one or more enzymatically active groups, and a polynucleotide tail which can be attached to the probe by annealing or hybridisation. Preferably the marker has a single-stranded polynucleotide tail complementary to that on the probe whereby the marker can be attached to the probe by a second hybridisation step. Presence of the sequence to be detected is confirmed by testing the labelled sample for any enzyme related activity.
Inventors
- 👤 Alan David Blair Malcolm
- 👤 Jean Lesley Nicolas
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 09:04
CPC Classifications
C; C07; C07H; C07H21/00; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/70
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
197 words
A method for the detection of a given nucleotide sequence in a polynucleotide, which comprises contacting the polynucleotide under the hybridisation conditions with a polynucleotide probe comprising a single-stranded nucleotide sequence complementary to the sequence to be detected and a reactive tail capable of undergoing coupling with an enzyme-labelled marker, coupling said marker to said tail, before or after hybridisation of the probe with said polynucleotide, assuming the presence therein of the given sequence, and determining the presence, or absence, of enzyme-associated activity in the sample after the said hybridisation with the probe and the said coupling of the marker to the probe, wherein the marker comprises a polynucleotide having a single-stranded portion to which the enzyme is attached by means of chemical linkages to one or more of the base groups in said single-stranded portion and a polynucleotide tail, the said probe likewise comprising a polynucleotide tail, the reactive tails of the probe and the marker both comprising nucleotide sequences differing from said complementary sequence and from the sequence to be detected, and wherein the probe is coupled to the marker by coupling the polynucleotide tail of the probe to the polynucleotide tail of the marker.
Claim 2
Independent Claim
197 words
A method for the detection of a given nucleotide sequence in a polynucleotide, which comprises contacting the polynucleotide under the hybridisation conditions with a polynucleotide probe comprising a single-stranded nucleotide sequence complementary to the sequence to be detected and a reactive tail capable of undergoing coupling with an enzyme-labelled marker, coupling said marker to said tail, before or after hybridisation of the probe with said polynucleotide, assuming the presence therein of the given sequence, and determining the presence, or absence, of enzyme-associated activity in the sample after the said hybridisation with the probe and the said coupling of the marker to the probe, wherein the marker comprises a polynucleotide having a single-stranded portion to which the enzyme is attached by means of chemical linkages to one or more of the base groups in said single-stranded portion and a polynucleotide tail, the said probe likewise comprising a polynucleotide tail, the reactive tails of the probe and the marker both comprising nucleotide sequences differing from said complementary sequence and from the sequence to be detected, and wherein the probe is coupled to the marker by coupling the polynucleotide tail of the probe to the polynucleotide tail of the marker.
Claim 3
Dependent Claim
62 words
A method according to claim 1, wherein the polynucleotide tails of the probe and the marker are both single-stranded nucleotide sequences complementary one to the other, but neither being complementary to the sequence to be detected, and wherein the coupling of the probe to the marker is effected by hybridisation of the tail of the marker to the tail of the probe.
Claim 4
Dependent Claim
26 words
A method according to claim 2, wherein the polynucleotide tails of the probe and the marker are both homogeneous nucleotide sequences complementary one to the other.
Claim 5
Dependent Claim
23 words
A method according to claim 3, wherein one of the polynucleotide tails is a poly(dA) tail and the other is a poly(dT) tail.
Claim 6
Independent Claim
50 words
A method according to any one of claims 1-4, wherein the marker comprises a glycoproteinaceous enzyme directly linked to the base groups of the single-stranded portion of the polynucleotide by means of chemical coupling between the glycollic groups of the glycoprotein to the amine groups of the nucleotide base groups.
Claim 7
Dependent Claim
12 words
A method according to claim 5, wherein the enzyme is horseradish peroxidase.
Claim 8
Dependent Claim
19 words
A method according to claim 6, wherein the marker is HRPO labelled, single-stranded, poly(dA) or poly(dT)-tailed phage M13 DNA.
Claim 9
Dependent Claim
27 words
An enzyme-containing polynucleotide marker comprising a single-stranded polynucleotide sequence having one or more enzymatically active groups chemically linked to the base groups of said single-stranded polynucleotide sequence.
Claim 10
Dependent Claim
27 words
An enzyme-containing polynucleotide marker comprising a single-stranded polynucleotide sequence having one or more enzymatically active groups chemically linked to the base groups of said single-stranded polynucleotide sequence.
Claim 11
Dependent Claim
11 words
A marker according to claim 8 further comprising a polynucleotide tail.
Claim 12
Dependent Claim
31 words
A marker according to claim 9, wherein said tail is a single-stranded polynucleotide tail capable of hybridisation with another polynucleotide possessing a single-stranded nucleotide sequence complementary to that of said tail.
Claim 13
Dependent Claim
19 words
A marker according to claim 10, wherein the polynucleotide tail consists of or comprises a homogeneous polynucleic acid sequence.
Claim 14
Dependent Claim
16 words
A marker according to claim 11, wherein the polynucleotide tail is a poly(dA) or poly(dT) tail.
Claim 15
Dependent Claim
52 words
A marker according to any one of claims 8-12, wherein the enzymatically active groups is or are derived from a glycoproteinaceous enzyme, and each group is attached to the single-stranded polynucleotide sequence by chemical bonds between glycollic groups in the glycoprotein and amine groups in the base groups of said polynucleotide sequence.
Claim 16
Dependent Claim
20 words
A marker according to any one of claims 8-13, wherein the enzymatically active groups is or are horseradish peroxidase groups.
Claim 17
Dependent Claim
8 words
HRPO-labelled, single-stranded, poly(dA) or poly(dT)-tailed phage M13 DNA.
Claim 18
Dependent Claim
8 words
HRPO-labelled, single-stranded, poly(dA) or poly(dT)-tailed phage M13 DNA.
Claim 19
Dependent Claim
93 words
A kit for the detection of a given nucleotide sequence in a polynucleotide sample comprising, as a first component, a polynucleotide probe comprising a single-stranded nucleotide sequence complementary to the sequence to be detected and a polynucleotide tail, and as a second component a marker comprising a polynucleotide having a single-stranded portion to the base groups of which is or are attached one or more enzymatically active groups, and a polynucleotide tail, the two tails being capable of coupling one to the other before or after hybridisation of the probe with the sample.
Claim 20
Dependent Claim
93 words
A kit for the detection of a given nucleotide sequence in a polynucleotide sample comprising, as a first component, a polynucleotide probe comprising a single-stranded nucleotide sequence complementary to the sequence to be detected and a polynucleotide tail, and as a second component a marker comprising a polynucleotide having a single-stranded portion to the base groups of which is or are attached one or more enzymatically active groups, and a polynucleotide tail, the two tails being capable of coupling one to the other before or after hybridisation of the probe with the sample.
Claim 21
Dependent Claim
29 words
A kit according to claim 16, wherein both the probe and the marker have single-stranded polynucleotide tails, the tail on the probe being complementary to that on the marker.
Claim 22
Dependent Claim
23 words
A kit according to claim 16 or 17, wherein the marker is an enzymatic marker as claimed in any one of claims 11-15.
References Cited (Backward Citations)
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