Methods and structures employing non-radioactive chemically-labeled polynucleotide probes

Claim 2

A substrate having fixed thereto a double-stranded polynucleotide, one of the strands of said double- stranded polynucleotide being a non-radioactive chemically labeled polynucleotide or comprises a non-radioactive chemically-labeled nucleotide as a nucleotide component of said one strand.

Claim 3

A substrate in accordance with Claim 1 wherein said substrate is a transparent substrate.

Claim 4

A substrate in accordance with Claim 1 wherein said chemically-labeled nucleotide is the compound, wherein B represents a purine, 7-deazapurine or pyrimidine moiety covalently bonded to the C 1 -position of the sugar moiety, provided that when B is purine or 7-deazapurine, it is attached at the N 9- position of the purine or deazapurine, and-when B is pyrimidine, it is attached at the N l- position; wherein A represents a moiety consisting of at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded ribonucleotide acid, deoxyribonucleic acid duplex, or DNA-RNA hybrid; wherein the dotted line represents a linkage or group joining B and A, provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; and wherein each of x, y and z represents

Claim 5

A substrate in accordance with Claim 1, wherein said chemically-labeled nucleotide has the formula, wherein each of B, B' and B" represents a purine, deazapurine, or pyrimidine moiety covalently bonded to the C l '-position of the sugar moiety, provided that whenever B, B' or B" is purine or deazapurine, it is attached at the N 9 -position of the purine or deazapurine, and whenever B, B' or B" is pyrimidine, it is attached at the Nl-position; wherein A represents a moiety consisting cf at least three carbon atoms which is capable of forming a detectable complex with a polypeptide when the compound is incorporated into a double-stranded duplex formed with a complementary ribonucleic or deoxyribonucleic acid molecule; wherein the dotted line represents a chemical linkage or group joining B and A, provided that if B is purine, the linkage is attached to the 8-position of the purine, if B is 7-deazapurine, the linkage is attached to the 7-position of the deazapurine, and if B is pyrimidine, the linkage is attached to the 5-position of the pyrimidine; wherein Z represents H- or HO-; and wherein m and n represent integers from 0 up to about 100,000.

Claim 6

A substrate in accordance with Claim I wherein said chemically-labeled nucleotide has the general formula, wherein P is the phosphoric acid moiety, S the sugar or monosaccharide moiety and B the base moiety, said nucleotide having covalently attached to the P or S or B moiety a chemical moiety Sig, said Sig chemical moiety when attached to the P moiety, is attached thereto via the chemical linkage, and when Sig is attached to the S moiety, the S moiety is a ribose group, said chemical moiety Sig when attached to said P, S or B being capable of signalling itself or making itself self-detecting or its presence known.

Claim 7

A method for detecting the presence of a pathogen in a clinical sample suspected of containing said pathogen, said method comprising depositing said sample on an inert transparent or translucent support, treating said sample to affix genetic material of any of said pathogens present in said sample to said support in substantially single-stranded form, contacting said fixed single-stranded genetic material with a non-radioactive chemically-labeled probe having a nucleotide sequence of at least about 25 bases at least substantially complementary to a nucleotide sequence of a structural gene characteristic of said pathogen, said chemically labeled probe having therein a chemically labeled nucleotide in accordance with Claim 5, said contacting being under hybridizing conditions at a predetermined stringency and detecting duplex formation on said support by means of said chemically labeled probe.

Claim 8

A substrate in accordance with Claim 1 wherein said substrate contains said double-stranded polynucleotide attached thereto, said double-stranded polynucleotide comprising said single-stranded non-radioactive chemically labeled polynucleotide or nucleotide, the other strand being unlabeled single-stranded polynucleotide, said substrate being prepared by fixing said other single-stranded unlabeled polynucleotide to said substrate and then hybridizing to said unlabled polynucleotide said one strand containing said chemically labeled polynucleotide or nucleotide.

Claim 9

A substrate in accordance with Claim 1 wherein said chemically labeled polynucleotide is a polynucleotide coupled to or attached to a polypeptide.

Claim 10

A substrate in accordance with Claim 1 wherein said chemically labeled polynucleotide is a polynucleotide coupled to or attached to a polypeptide, said polypeptide being terminally attached to or ligated to said polynucleotide.

Claim 11

A substrate in accordance with Claim 1 wherein said chemically labeled polynucleotide comprises or has attached thereto an amino acid or polypeptide comprising a Sic chemical moiety covalently attached thereto, said Sig chemical moiety being capable of signalling itself or making itself self-detecting or its presence known.

Claim 12

A substrate in accordance with Claim 10 wherein said Sig chemical moiety comprises a saccharide component.

Claim 13

A substrate in accordance with Claim 1 wherein said chemically labeled polynucleotide comprises or has attached thereto a monosaccharide or a polysaccharide comprising a Sig chemical moiety attached thereto, said S ig chemical moiety being capable of signalling itself or making itself self-protecting or its presence known.

Claim 14

A substrate in accordance with Claim 27 wherein said Sig chemical moiety comprises a chelating agent.

Claim 15

A substrate in accordance with Claim 1 wherein said substrate is transparent and wherein said substrate is adapted for the transmission of light therethrough for color observation or colorimetric determination of said double-stranded polynucleotide on said transparent substrate.

Claim 16

A substrate in accordance with Claim 1 wherein said substrate is a planar substrate provided with a well therein wherein said double-stranded polynucleotide is fixed.

Claim 17

A substrate in accordance with Claim 15 wherein said substrate carrying said double-stranded polynucleotide fixed to a well therein is adapted for the transmission of light therethrough for the photometric or colorimetric determination of the double-stranded polynucleotide fixed within said well.

Claim 20

18 . A method in accordance with Claim 52 wherein said surface is a transparent surface.

Claim 21

A method in accordance with Claim 17 wherein said surface is a glass surface and wherein said single- stranded genetic material is fixed to a well provided on said surface, said surface being adapted for the transmission of light therethrough for the photometric or colorimetric determination of duplex formation between said single-stranded genetic material and said polynucleotide probe.

Claim 22

A method in accordance with Claim 52 wherein duplex or double strand formation or hybridization between said single strand of material and said non-radioactive chemically-labeled polynucleotide probe is determined by providing said chemical label attached to said polynucleo- tiode probe, before or after duplex formation, with a chelating agent and eliciting the presence of said chelating agent by contacting said chelating agent with a substrate which undergoes a chemical reaction or color change when in contact with said chelating agent.

Claim 23

A method in accordance with Claim 20 wherein eliciting the presence of said chelating agent is carried out by contacting said chelating agent with a substrate which undergoes a chemical reaction when in contact with said chelating agent.

Claim 24

A method in accordance with Claim 2 0 wherein eliciting the presence of said chelating agent is carried out by contacting said chelating agent with a substrate which undergoes a color change when in contact with said chelating agent.

Claim 27

A transparent glass surface suitable for fixing genetic DNA material thereto, said glass surface having attached or fixed thereto amino groups.

Claim 28

A transparent glass surface suitable for fixing genetic DNA material thereto, said glass surface having attached or fixed thereto amino groups.

Claim 29

A transparent-glass substrate, the surface of which has been treated in accordance with Claim 23.

Claim 30

Z6. A method of preparing a surface selected from the group consisting of a glass surface or a plastic surface for the fixing of genetic material thereto, which comprises contacting said surface with the amine and the hardener components of an epoxy glue to form a coating of epoxy glue thereon.

Claim 31

Z6. A method of preparing a surface selected from the group consisting of a glass surface or a plastic surface for the fixing of genetic material thereto, which comprises contacting said surface with the amine and the hardener components of an epoxy glue to form a coating of epoxy glue thereon.

Claim 34

-28. A planar form transparent glass plate provided with rows of wells or depressions formed on the surface thereof, the surface of said wells or depressions having fixed thereto double-stranded polynucleotide, one of the strands of said double-stranded nucleotide being a non-radioactively chemically-labeled polynucleotide or comprises a non-radioactively chemically-labeled nucleotide as a nucleotide component of said one strand.

Claim 35

-28. A planar form transparent glass plate provided with rows of wells or depressions formed on the surface thereof, the surface of said wells or depressions having fixed thereto double-stranded polynucleotide, one of the strands of said double-stranded nucleotide being a non-radioactively chemically-labeled polynucleotide or comprises a non-radioactively chemically-labeled nucleotide as a nucleotide component of said one strand.

Claim 36

A transparent glass container or cuvette provided with planar walls, the interior surface of the container having fixed thereto double-stranded polynucleotide, one of the strands of said double-stranded polynucleotide being a non-radioactively chemically-labeled polynucleotide or comprises a non-radioactively chemically-labeled nucleotide as a nucleotide component of said one-strand.

Claim 37

A transparent glass container or cuvette provided with planar walls, the interior surface of the container having fixed thereto double-stranded polynucleotide, one of the strands of said double-stranded polynucleotide being a non-radioactively chemically-labeled polynucleotide or comprises a non-radioactively chemically-labeled nucleotide as a nucleotide component of said one-strand.

Claim 38

A method for detecting the presence or identity of genetic DNA material which comprises depositing a sample of genetic material on an inert transparent or trans- leucent support surface, treating said sample to affix said genetic DNA material to said support in substantially single-stranded form, contacting said fixed single-stranded genetic DNA material with a non-radioactive chemically-labeled probe having a nucleotide sequence of at least about 25 bases, at least substantially complementary to the nucleotide sequence contained in said genetic DNA material, said chemically-labeled probe having therein a chemically- labled nucleotide in accordance with Claim 3, said contacting being under hybridizing conditions at a predetermined stringency and detecting duplex formation of said genetic DNA material fixed to said support surface by means of said chemically-labeled probe.

Claim 39

-- 3 1. A substrate in accordance with Claim 1 wherein said substrate is selected from the group consisting of glass, glass coated material, plastic, plastic coated material,' siliceous material, cellulose, nitrocellulose or dextran and wherein the chemical label moiety of said non-radioactive chemically labeled nucleotide comprises an enzyme component effective upon contact with a chromogen to produce an insoluble colored precipitate or product so as to indicate, define or elicit the presence and/or location of said double-stranded polynucleotide on said substrate. --

Claim 40

-- 32. A method for detecting or eliciting the presence of a pathogen in a clinical sample suspected of containing said pathogen, said method comprising depositing said sample on a substrate, treating said sample to affix genetic material of any of said pathogens present in said sample to said substrate in substantially single-stranded form, contacting said fixed single-stranded genetic material with a non-radioactive chemically labeled polynucleotide probe having a nucleotide sequence substantially complementary to the nucleotide sequence of said single-stranded genetic material affixed to said substrate, said contacting being carried out under hybridizing conditions at a predetermined-stringency to effect duplex formation between said single-stranded genetic material and said non-radioactive chemically labeled polynucleotide probe, adding or attaching to the chemical label moiety of said non-radioactive chemically labeled polynucleotide probe a complex comprising an enzyme component - effective upon contact with a chromogen to produce an insoluble colored precipitate or product and bringing the resulting formed duplex comprising said chemically labeled polynucleotide probe and said single stranded genetic material into contact with said chromogen to produce said insoluble colored precipitate or product, thereby detecting or eliciting or defining on said substrate the presence of said single-stranded genetic material derived from said pathogen.--

Claim 41

-- 32. A method for detecting or eliciting the presence of a pathogen in a clinical sample suspected of containing said pathogen, said method comprising depositing said sample on a substrate, treating said sample to affix genetic material of any of said pathogens present in said sample to said substrate in substantially single-stranded form, contacting said fixed single-stranded genetic material with a non-radioactive chemically labeled polynucleotide probe having a nucleotide sequence substantially complementary to the nucleotide sequence of said single-stranded genetic material affixed to said substrate, said contacting being carried out under hybridizing conditions at a predetermined-stringency to effect duplex formation between said single-stranded genetic material and said non-radioactive chemically labeled polynucleotide probe, adding or attaching to the chemical label moiety of said non-radioactive chemically labeled polynucleotide probe a complex comprising an enzyme component - effective upon contact with a chromogen to produce an insoluble colored precipitate or product and bringing the resulting formed duplex comprising said chemically labeled polynucleotide probe and said single stranded genetic material into contact with said chromogen to produce said insoluble colored precipitate or product, thereby detecting or eliciting or defining on said substrate the presence of said single-stranded genetic material derived from said pathogen.--

Claim 42

-- 33. A method for determining the presence of a selected single-stranded genetic material which has been fixed to a substrate, which comprises bringing said fixed single-stranded genetic material into contact under hybridizing conditions with chemically labeled single-stranded polynucleotide probe having a nucleotide sequence sufficiently complementary with the nucleotide sequence characterizing said selected single-stranded genetic material to effect hybridization of said single-stranded polynucleotide probe with said single-stranded genetic material, adding to the chemical label moiety of said chemically-labeled polynucleotide probe for attachment thereto a chemical component effective on contact with a chromogen to produce an insoluble colored precipitate or product and bringing the resulting formed hybrid comprising said chemically-labeled polynucleotide probe and said single-stranded genetic material into contact with said chromogen to produce said insoluble colored precipitate or product, thereby determining or eliciting or defining on said substrate the presence of said selected single-stranded genetic material. --

Claim 43

-- 33. A method for determining the presence of a selected single-stranded genetic material which has been fixed to a substrate, which comprises bringing said fixed single-stranded genetic material into contact under hybridizing conditions with chemically labeled single-stranded polynucleotide probe having a nucleotide sequence sufficiently complementary with the nucleotide sequence characterizing said selected single-stranded genetic material to effect hybridization of said single-stranded polynucleotide probe with said single-stranded genetic material, adding to the chemical label moiety of said chemically-labeled polynucleotide probe for attachment thereto a chemical component effective on contact with a chromogen to produce an insoluble colored precipitate or product and bringing the resulting formed hybrid comprising said chemically-labeled polynucleotide probe and said single-stranded genetic material into contact with said chromogen to produce said insoluble colored precipitate or product, thereby determining or eliciting or defining on said substrate the presence of said selected single-stranded genetic material. --

Claim 46

35 .' A material, device or system for detecting or measuring a color change or chemical reaction colorimetrically or photometrically which is packaged in association with instructions for use in accordance with the method of Claim 6.

Claim 47

36 . A transparent substrate prepared in accordance with Claim 26 packaged in association with instructions for use in accordance with Claim 30..

Claim 48

37 . A transparent substrate prepared in accordance with Claim 66 useful in the device or system of Claim 34 for detecting or measuring a color change or chemical reaction colorimetrically or photometrically packaged in association with instructions for use in the device or system of Claim 34