EP0133473A2 — In vivo labelling of polynucleotide sequences

Claim 1

Independent Claim 60 words

. A process for in vivo labelling of polynucleotide sequences comprising the step of culturing a host characterized by a polynucleotide sequence (a) that is desired to be labelled and at 'least another polynucleotide sequence (b) that expresses a product on culturing of the host that labels the desired polynucleotide sequence (a) when it is replicated in the cultured host.

Claim 2

Dependent Claim 60 words

. A process for in vivo labelling of polynucleotide sequences comprising the step of culturing a host characterized by a polynucleotide sequence (a) that is desired to be labelled and at 'least another polynucleotide sequence (b) that expresses a product on culturing of the host that labels the desired polynucleotide sequence (a) when it is replicated in the cultured host.

Claim 3

Dependent Claim 60 words

.The process of claim 1, wherein polynucleotide sequence (a) is selected from the group consisting of a DNA sequence that is part of the native genome of said host, a DNA sequence that is inserted into the native genome of said host and DNA sequences that comprise at least a part of a DNA sequence employed to transfect said host..

Claim 4

Dependent Claim 46 words

The process of claim 2, wherein the DNA sequence employed to transfect said host is selected from the group consisting of cloning vehicles and phages characterized by the polynucleotide sequence desired to be labelled, said cloning vehicles and phages being capable of replication in the host.

Claim 5

Dependent Claim 58 words

The process of claim 1, wherein polynucleotide sequence (b) is selected from the group consisting of a DNA sequence that is part of the native genome of said host, a DNA sequence that is inserted into the native genome of said host and DNA sequences that comprise at'least part of a DNA sequence employed to transfect said host.

Claim 6

Dependent Claim 64 words

The process of claim 4, wherein the DNA sequence employed to transfect said host is selected from the group consisting of cloning vehicles and phages characterized by the polynucleotide sequence that expresses a product on culturing of the host that labels the desired polynucleotide sequence on its replication in the cultured host, said cloning vehicles and phages being capable of replication in the host.

Claim 7

Dependent Claim 23 words

The process of claim 1, wherein said polynucleotide sequence (b) is derived from the phages that label polynucleotide sequences contained in that phage.

Claim 8

Dependent Claim 19 words

The process of claim 6, wherein said phages are selected from the group consisting of the T-even E.coli bacteriophages.

Claim 9

Dependent Claim 22 words

'The process of claim 7, wherein said phages are selected from the group consisting of bacteriophage T2, bacteriophage T4 and bacteriophage T6.

Claim 10

Dependent Claim 18 words

The process of claim 1, wherein said polynucleotide sequence (b) is derived from the Xanthomonas oryzae bacteriophage XP12.

Claim 11

Dependent Claim 17 words

The process of claim 1, wherein said polynucleotide sequence (b) is derived from the Bacillus phage SPO1.

Claim 12

Dependent Claim 26 words

The process of claim 1, wherein said label is carried by a compound selected from the group consisting of glycosylated nucleotides, glucosylated nucleotides, deoxyhydroxymethyluridine and 5-methylcytosine.

Claim 13

Dependent Claim 28 words

The process of claim 1 further comprising the step of isolating from said cultured host a DNA sequence comprising at least the polynucleotide sequence desired to be labelled.

Claim 14

Dependent Claim 22 words

The process of claim 12, wherein the isolated DNA sequence is part or all of the polynucleotide sequence desired to be labelled.

Claim 15

Dependent Claim 65 words

.A process for in vivo labelling of polynucleotide sequences comprising the step of culturing a host containing a polynucleotide sequence that is desired to be labelled, in the presence of a base, nucleoside or nucleotide, or analogue or precursor thereof, that the host or a variant thereof requires for its growth, said base, nucleoside or nucleotide, or analogue or precursor thereof, carrying the desired label.

Claim 16

Dependent Claim 65 words

.A process for in vivo labelling of polynucleotide sequences comprising the step of culturing a host containing a polynucleotide sequence that is desired to be labelled, in the presence of a base, nucleoside or nucleotide, or analogue or precursor thereof, that the host or a variant thereof requires for its growth, said base, nucleoside or nucleotide, or analogue or precursor thereof, carrying the desired label.

Claim 17

Dependent Claim 64 words

The process of claim 14, wherein said polynucleotide sequence that is desired to be labelled is selected from the group consisting of DNA sequences that are part of the native genome of the host, DNA sequences that are inserted into the native genome of the host, and DNA sequences that comprise at least a part of a DNA sequence employed to transfect said host.

Claim 18

Dependent Claim 46 words

The process of claim 15, wherein said DNA sequence employed to transfect said host is selected from the group of cloning vehicles and phages characterized by the polynucleotide sequence that is desired to be labelled, said vehicles and phages being capable of replication in said host.

Claim 19

Dependent Claim 27 words

The process of claim 14, wherein said host is selected from the group consisting of thymine or thymidine minus mutants and the label carrying analogue is BrdUR.

Claim 20

Dependent Claim 38 words

The process of claim 1 or 14 further comprising the step of attaching or binding a signalling moiety, a bridging moiety or at least one combination of bridging moiety and signalling moiety to said label of the polynucleotide'sequence.

Claim 21

Dependent Claim 27 words

'19. The process of claim 18, wherein said signalling moiety , bridging moiety or combination of bridging moiety and signalling moiety is covalently bound to said label.

Claim 22

Dependent Claim 47 words

The process of cla:.m 19, wherein said signalling moiety and bridging moiety is selected from the group consisting of radioactive compounds, biotin, fluorescent compounds, fluorescein, rhodamine, dansyl, magnetic compounds, chelating agent.s and ; other signalling and bridging moieties which may be covalently attached to labelled polynucleotide sequences.

Claim 23

Dependent Claim 47 words

The process of cla:.m 19, wherein said signalling moiety and bridging moiety is selected from the group consisting of radioactive compounds, biotin, fluorescent compounds, fluorescein, rhodamine, dansyl, magnetic compounds, chelating agent.s and ; other signalling and bridging moieties which may be covalently attached to labelled polynucleotide sequences.

Claim 24

Dependent Claim 25 words

The process of claim 18, wherein said signalling moiety, bridging moiety or combination of bridging moiety and signalling moiety is non-covalently bound to said label.

Claim 25

Dependent Claim 53 words

The process of claim 21, wherein said signalling moiety and bridging moiety is selected from the group consisting of polypeptides, proteins, lectins, Concanavalin A, enzymes, alkaline phosphatases, acid phosphatases, antigens, antibodies, β-galactosidase, glucose oxidase, horseradish peroxidase, chelating agents and other signalling and bridging moieties which may be non-covalently bound to labelled polynucleotide sequences.

Claim 26

Dependent Claim 17 words

A polynucleotide sequence that is labelled in accordance with the process of any one of claims 1-17.

Claim 27

Dependent Claim 20 words

The polynucleotide sequence of claim 23, characterized in that it is selected from the group consisting of DNA and RNA.

Claim 28

Dependent Claim 55 words

The polynucleotide sequence of claim 23, characterized in that said label on the polynucleotide sequence is further attached or bound to a signalling moiety, a bridging moiety or at least one combination of bridging moiety and signalling moiety, said signalling moiety, bridging moiety, or combination not interfering with the hybridization of said labelled polynucleotide sequence.

Claim 29

Dependent Claim 28 words

- The polynucleotide sequence of claim 25., wherein said signalling moiety, bridging moiety or said combination of bridging moiety and signalling moiety is covalently bound to said label.

Claim 30

Dependent Claim 45 words

The polynucleotide sequence of claim 26, wherein said signalling and bridging moiety is selected from the group consisting of radioactive compounds; fluorescent compounds, biotin, fluorescein, rhodamine, dansyl, magnetic compounds, chelating agents and other signalling and bridging moieties which may be covalently attached to said label.

Claim 31

Dependent Claim 28 words

'28. The polynucleotide sequence of claim 25, wherein said signalling moiety, bridging moiety or said combination of signalling moiety and bridging moiety is non-covalently bound to said label.

Claim 32

Dependent Claim 53 words

The polynucleotide sequence of claim 28, wherein said signalling moiety and bridging moiety is selected from the group consisting of polypeptides, proteins, lectins, Concanavalin A, enzymes, alkaline phosphatase, acid phosphatase, antigens, antibodies, β-galactosidase, glucose oxidase, horseradish peroxidase, chelating agents and other signalling and bridging moieties which may be non-covalently attached to said label.

Claim 33

Dependent Claim 14 words

A detection kit for analytes characterized by a polynucleotide sequence according to claim 23.

Claim 34

Dependent Claim 15 words

A detection kit for analytes characterized by a polynucleotide sequence according to claim 25. .

Claim 35

Dependent Claim 41 words

' A method for detecting a polynucleotide sequence in an analyte comprising the steps of hybridizing said analyte to the labelled polynucleotide sequence of claim 23 that :.s complementary to the desired polynucleotide sequence in the analyte' and detecting that hybridization.

Claim 36

Dependent Claim 46 words

The method of claim 32, comprising the further step of attaching c:: binding to the label of the polynucleotide sequence iifter hybridization of that sequence to the complementary sequence in the analyte a signalling moiety or at least one combination of bridging moiety and signalling moiety.

Claim 37

Dependent Claim 25 words

The method of claim 33, wherein said signalling or said combination of bridging moiety and signalling moiety is attached covalently or non-covalently to said label.

Claim 38

Dependent Claim 40 words

A method for detecting a polynucleotide sequence in an analyte comprising the steps of hybridizing said analyte to a labelled polynucleotide sequence of claim 25 that is complementary to the desired polynucleotide sequence in the analyte and detecting that hybridization.

In vivo labelling of polynucleotide sequences

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