US4617261A — Process for labeling nucleic acids and hybridizat…

Claim 1

Independent Claim 140 words

A process for labeling a partially double-stranded nucleic acid which comprises the steps of: (a) contacting the nucleic acid with one or more labeling compositions of the formula: [A--[B--L wherein A is an alkylating intercalation moiety, B is a divalent organic moiety having the formula: ##STR23## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, and L is a monovalent label moiety, wherein B is exclusive of any portion of the intercalation and label moieties, said contacting occurring so as to cause the alkylating intercalation moiety of the labeling composition to intercalate into the nucleic acid to form a complex; and (b) activating the complex so as to induce the alkylating intercalation moiety to bond covalenty to at least one of the nucleic acid strands.

Claim 2

Dependent Claim 140 words

A process for labeling a partially double-stranded nucleic acid which comprises the steps of: (a) contacting the nucleic acid with one or more labeling compositions of the formula: [A--[B--L wherein A is an alkylating intercalation moiety, B is a divalent organic moiety having the formula: ##STR23## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, and L is a monovalent label moiety, wherein B is exclusive of any portion of the intercalation and label moieties, said contacting occurring so as to cause the alkylating intercalation moiety of the labeling composition to intercalate into the nucleic acid to form a complex; and (b) activating the complex so as to induce the alkylating intercalation moiety to bond covalenty to at least one of the nucleic acid strands.

Claim 3

Dependent Claim 17 words

The process of claim 1 wherein the nucleic acid is DNA and the label moiety is non-radioactive.

Claim 4

Dependent Claim 17 words

The process of claim 1 wherein step (b) results in covalent bonding to both nucleic acid strands.

Claim 5

Dependent Claim 12 words

The process of claim 1 wherein A is of the formula: ##STR24##

Claim 6

Dependent Claim 172 words

A process for preparing a labeled nucleic acid hybridization probe for detecting nucleic acid sequences which process comprises the steps of: (a) contacting a nucleic acid comprising a double-stranded nucleic acid region adjacent to a single-stranded region capable of hybridization with the nucleic acid sequence to be detected, with at least one of the labeling compositions of the formula: [A--[B--L wherein A is an alkylating intercalation moiety, B is a divalent organic moiety having the formula: ##STR25## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, and L is a monovalent label moiety, wherein B is exclusive of any portion of the intercalation and label moieties, said contacting causing the alkylating intercalation moiety of the labeling composition(s) to intercalate into the nucleic acid to form a complex; and (b) activating the complex to induce the alkylating intercalation moiety to bond covalently to the double-stranded nucleic acid region, via either one or both of the nucleic acid strands.

Claim 7

Dependent Claim 172 words

A process for preparing a labeled nucleic acid hybridization probe for detecting nucleic acid sequences which process comprises the steps of: (a) contacting a nucleic acid comprising a double-stranded nucleic acid region adjacent to a single-stranded region capable of hybridization with the nucleic acid sequence to be detected, with at least one of the labeling compositions of the formula: [A--[B--L wherein A is an alkylating intercalation moiety, B is a divalent organic moiety having the formula: ##STR25## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, and L is a monovalent label moiety, wherein B is exclusive of any portion of the intercalation and label moieties, said contacting causing the alkylating intercalation moiety of the labeling composition(s) to intercalate into the nucleic acid to form a complex; and (b) activating the complex to induce the alkylating intercalation moiety to bond covalently to the double-stranded nucleic acid region, via either one or both of the nucleic acid strands.

Claim 8

Dependent Claim 31 words

The process of claim 5 wherein A of the labeling compound is of the formula: ##STR26## and the activating step (b) is carried out by irradiating the complex with ultraviolet light.

Claim 9

Dependent Claim 25 words

The process of claim 5 wherein the nucleic acid is DNA and L is detectable by spectroscopy, photochemistry, or by biochemical, immunochemical or chemical means.

Claim 10

Dependent Claim 41 words

The process of claim 5 wherein step (a) is carried out by incubating the nucleic acid with the labeling composition in a medium at about 0° to 50° C. containing a buffer with a pH of between about 6 and 9.

Claim 11

Dependent Claim 24 words

The process of claim 8 wherein the medium is at about 4° to 20° C. and the pH is between about 6 to 8.

Claim 12

Dependent Claim 22 words

The process of claim 6 wherein step (b) is carried out using ultraviolet light of from about 350 to 390 nm wavelength.

Claim 13

Dependent Claim 34 words

The process of claim 10 wherein the wavelength is about 360 nm and the step is carried out at from 1 to 100 mWatts per cm 2 for from one minute to 24 hours.

Claim 14

Dependent Claim 21 words

The process of claim 5 wherein the alkylating intercalation moiety bonds covalently to both strands of the double-stranded nucleic acid region.

Claim 15

Dependent Claim 254 words

A process for preparing a labeled DNA hybridization probe for detecting DNA sequences which process comprises the steps of: (a) inserting a DNA fragment which contains a sequence complementary to the sequence to be detected into the bacteriophage M13 genome at a restriction site therein and preparing therefrom circular single-stranded DNA containing the DNA fragment; (b) cleaving double-stranded M13 DNA at a restriction site which is less than 100 base pairs from, or is the same as, the restriction site used to insert the DNA fragment in step (a); (c) denaturing the cleaved double-stranded M13 DNA; (d) hybridizing the circular single-stranded M13 phage isolated from step (a) with the denatured double-stranded M13 DNA; (e) removing any single-stranded DNA from the mixture to obtain a circular partially double-stranded DNA; (f) contacting the DNA from step (e) with at least one labeling composition of the formula: [A--[B--L wherein A is an alkylating intercalation moiety, B is a divalent organic moiety having the formula: ##STR27## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, and L is a monovalent label moiety, wherein B is exclusive of any portion of the intercalation and label moieties, said contacting causing the alkylating intercalation moiety of the labeling composition(s) to intercalate into the DNA to form a complex; and (g) activating the complex to induce the alkylating intercalation moiety to bond covalently to the double-stranded DNA region, via either one or both of the DNA strands.

Claim 16

Dependent Claim 254 words

A process for preparing a labeled DNA hybridization probe for detecting DNA sequences which process comprises the steps of: (a) inserting a DNA fragment which contains a sequence complementary to the sequence to be detected into the bacteriophage M13 genome at a restriction site therein and preparing therefrom circular single-stranded DNA containing the DNA fragment; (b) cleaving double-stranded M13 DNA at a restriction site which is less than 100 base pairs from, or is the same as, the restriction site used to insert the DNA fragment in step (a); (c) denaturing the cleaved double-stranded M13 DNA; (d) hybridizing the circular single-stranded M13 phage isolated from step (a) with the denatured double-stranded M13 DNA; (e) removing any single-stranded DNA from the mixture to obtain a circular partially double-stranded DNA; (f) contacting the DNA from step (e) with at least one labeling composition of the formula: [A--[B--L wherein A is an alkylating intercalation moiety, B is a divalent organic moiety having the formula: ##STR27## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, and L is a monovalent label moiety, wherein B is exclusive of any portion of the intercalation and label moieties, said contacting causing the alkylating intercalation moiety of the labeling composition(s) to intercalate into the DNA to form a complex; and (g) activating the complex to induce the alkylating intercalation moiety to bond covalently to the double-stranded DNA region, via either one or both of the DNA strands.

Claim 17

Dependent Claim 51 words

The process of claim 13 wherein A of the labeling composition is of the formula: ##STR28## the label moiety is non-radioactive, the activating step (g) is carried out by irradiating the complex with ultraviolet light, and the alkylating intercalation moiety bonds covalently to both strands of the double-stranded nucleic acid region.

Claim 18

Dependent Claim 14 words

The process of claim 14 wherin the labeling composition is of the formula: ##STR29##

Claim 19

Dependent Claim 14 words

The process of claim 14 wherein the labeling composition is of the formula: ##STR30##

Claim 20

Dependent Claim 123 words

A labeled nucleic acid hybridization probe for detecting nucleic acid sequences comprising a double-stranded nucleic acid region adjacent to a single-stranded region capable of hybridization with the nucleic acid sequence to be detected, said double-stranded nucleic acid region being covalently bound to at least one alkylating intercalation moiety via either one or both of the nucleic acid strands, said moiety being bound to a divalent organic moiety having the formula: ##STR31## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, which organic moiety is in turn bound to a monovalent label moiety, wherein said divalent organic moiety is exclusive of any portion of the intercalation and label moieties.

Claim 21

Dependent Claim 123 words

A labeled nucleic acid hybridization probe for detecting nucleic acid sequences comprising a double-stranded nucleic acid region adjacent to a single-stranded region capable of hybridization with the nucleic acid sequence to be detected, said double-stranded nucleic acid region being covalently bound to at least one alkylating intercalation moiety via either one or both of the nucleic acid strands, said moiety being bound to a divalent organic moiety having the formula: ##STR31## where Y is O, NH or N--CHO, x is a number from 1 to 4 and y is a number from 2 to 4, which organic moiety is in turn bound to a monovalent label moiety, wherein said divalent organic moiety is exclusive of any portion of the intercalation and label moieties.

Claim 22

Dependent Claim 24 words

The probe of claim 17 wherein the double-stranded nucleic acid is circular and the single-stranded region is an insert in one of the strands.

Claim 23

Dependent Claim 14 words

The probe of claim 17 wherein the alkylating intercalation moiety is a 4'-methylene-substituted-4,5',8-trimethylpsoralen moiety.

Claim 24

Dependent Claim 39 words

The probe of claim 17 wherein the label moiety is detectable by spectroscopy or photochemistry or by formation of a detectable complex between the label moiety and a polypeptide, lectin or antibody with or without an enzyme associated therewith.

Claim 25

Dependent Claim 25 words

The probe of claim 19 wherein the bound conjugate of divalent organic moiety and monovalent label moiety has the formula: ##STR32## wherein Y is N--CHO.

Claim 26

Dependent Claim 11 words

The probe of claim 17 wherein the nucleic acid is DNA.

Claim 27

Dependent Claim 19 words

The probe of claim 17 wherein the label moiety is detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means.

Claim 28

Dependent Claim 25 words

The probe of claim 17 wherein the nucleic acid region is covalently bound to said alkylating intercalation moiety via both of the nucleic acid strands.

Claim 29

Dependent Claim 19 words

The probe of claim 17 wherein the nucleic acid sequence to be detected is characteristic of a pathogenic microbe.

Claim 30

Dependent Claim 28 words

The probe of claim 17 wherein the sequence to be detected is in a gene involved in HLA typing or is associated with a genetic or infectious disease.

Claim 31

Dependent Claim 22 words

A complex comprising the probe of claim 26 and a polypeptide, lectin, or antibody capable of forming a complex with said probe.

Claim 32

Dependent Claim 38 words

The complex of claim 27 wherein the polypeptide, lectin or antibody includes a detectable moiety selected from the group consisting of a fluorescent dye, an electron dense reagent, or an enzyme capable of depositing an insoluble reaction product.

Claim 33

Dependent Claim 22 words

A complex comprising the probe of claim 26 containing biotin as the label moiety and a conjugate of streptavidin and a peroxidase.

Claim 34

Dependent Claim 141 words

A process for detecting the presence of one or more nucleic acid sequences in a sample suspected of containing the sequence or sequences comprising: (a) contacting the sample with an effective amount of reagent sufficient to open the cells, body fluid, viral capsids, or tissue of the sample and to separate the strands of the nucleic acid or acids; (b) depositing the sample before, during, or after step (a) on an inert support; (c) contacting the deposited sample with an effective amount of reagent sufficient to affix a substantially single-stranded form of the nucleic acid or acids on the support; (d) contacting the affixed nucleic acid single-stranded form with an effective amount of the hybridization probe of claim 17 under hybridization conditions; and (e) detecting hybridization of the single-stranded nucleic acid sequences by means of the label moiety on the probe.

Claim 35

Dependent Claim 26 words

The process of claim 30 wherein the sequence or sequences to be detected are characteristic of a pathogenic microbe or are associated with an infectious disease.

Claim 36

Dependent Claim 237 words

A process for conducting HLA typing comprising: (a) digesting genomic HLA DNA from an individual with a restriction endonuclease which produces a polymorphic digestion pattern with HLA DNA; (b) subjecting the digestion products to gel electrophoresis; (c) transferring the sample from step (b) to a filter; (d) contacting the sample with an effective amount of a base sufficient to expose the nucleic acids in the sample and to separate the strands of the nucleic acid or acids in said sample; (e) neutralizing said sample; (f) exposing said sample to UV light, a drying solvent, or heat to affix a substantially single-stranded form of the nucleic acid or acids in the sample on the filter at about the same site on the filter where the sample was deposited; (g) contacting said affixed single-stranded DNA sample with an effective amount of a reagent which prevents nonspecific reaction of the filter with the probe to be employed; (h) contacting said affixed single-stranded DNA sample with an effective amount of the hybridization probe of claim 21 containing a single-stranded HLA gene as the hybridizing region under hybridization conditions; (i) washing said sample of unhybridized probe; (j) detecting hybridization of the single-stranded nucleic acid sequence or sequences in the sample by means of the label moiety on the probe; and (k) comparing the detected products from step (j) with the restriction fragment pattern of a known sample and/or with molecular weight markers.

Claim 37

Dependent Claim 36 words

The process of claim 32 wherein step (j) is accomplished by contacting the probe with a conjugate of streptavidin and horseradish peroxidase, adding a peroxidase substrate and detecting visually the reaction product of peroxidase and substrate.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1989-10-13	1990-04-14	1990-10-14	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1993-10-13	1994-04-14	1994-10-14	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	1997-10-13	1998-04-14	1998-10-14	$7700.00	Window Closed / Lapsed

Process for labeling nucleic acids and hybridization probes

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