Process for labeling single-stranded nucleic acids and hybridizaiton probes

Claim 2

A process for labeling a single-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 moiety, B is a divalent organic moiety having the formula: ##STR10## where Y is 0, 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 alkylating and label moieties, said contacting occurring so as to cause the alkylating moiety of the labeling composition to complex with the nucleic acid; and (b) activating the complex so as to induce the alkylating moiety to bond covalently to the nucleic acid.

Claim 3

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

Claim 4

The process of claim 1 wherein A is of the formula: ##STR11##

Claim 5

A process for preparing a labeled single-stranded nucleic acid hybridization probe for detecting homologous nucleic acid sequences which process comprises the steps of: (a) contacting a single-stranded nucleic acid comprising 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 moiety, B is a divalent organic moiety having the formula: ##STR12## where Y is 0, 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 alkylating and label moieties, said contacting causing the alkylating moiety of the labeling composition(s) to complex with the nucleic acid; and (b) activating the complex to induce the alkylating moiety to bond covalently to the nucleic acid.

Claim 6

A process for preparing a labeled single-stranded nucleic acid hybridization probe for detecting homologous nucleic acid sequences which process comprises the steps of: (a) contacting a single-stranded nucleic acid comprising 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 moiety, B is a divalent organic moiety having the formula: ##STR12## where Y is 0, 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 alkylating and label moieties, said contacting causing the alkylating moiety of the labeling composition(s) to complex with the nucleic acid; and (b) activating the complex to induce the alkylating moiety to bond covalently to the nucleic acid.

Claim 7

The process of claim 4 wherein A of the labeling compound is of the formula: ##STR13## and the activating step (b) is carried out by irradiating the complex with ultraviolet light.

Claim 8

The process of claim 4 wherein the nucleic acid is DNA and L is detectable by spectroscopy, photochemistry, or by biochemical, immunochemical or chemical means.

Claim 9

The process of claim 4 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 10

The process of claim 7 wherein the medium is at about 4° to 20° C. and the pH is between about 6 and 8.

Claim 11

The process of claim 5 wherein step (b) is carried out using ultraviolet light of from about 350 to 390 nm wavelength.

Claim 12

The process of claim 9 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 15 hours.

Claim 13

A process for preparing a labeled single-stranded DNA hybridization probe for detecting homologous 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) contacting the DNA from step (a) with at least one labeling composition of the formula: [A--[B--L wherein A is an alkylating moiety, B is a divalent organic moiety having the formula: ##STR14## where Y is 0, 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 alkylating and label moieties, said contacting causing the alkylating moiety of the labeling composition(s) to complex with the DNA; and (c) activating the complex to induce the alkylating moiety to bond covalently to the single-stranded DNA.

Claim 14

A process for preparing a labeled single-stranded DNA hybridization probe for detecting homologous 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) contacting the DNA from step (a) with at least one labeling composition of the formula: [A--[B--L wherein A is an alkylating moiety, B is a divalent organic moiety having the formula: ##STR14## where Y is 0, 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 alkylating and label moieties, said contacting causing the alkylating moiety of the labeling composition(s) to complex with the DNA; and (c) activating the complex to induce the alkylating moiety to bond covalently to the single-stranded DNA.

Claim 15

The process of claim 11 wherein A of the labeling composition is of the formula: ##STR15## the label moiety is non-radioactive, and the activating step (c) is carried out by irradiating the complex with ultraviolet light.

Claim 16

The process of claim 12 wherein the labeling composition is of the formula: ##STR16##

Claim 17

The process of claim 12 wherein the labeling composition is of the formula: ##STR17##

Claim 18

A labeled single-stranded nucleic acid hybridization probe for detecting homologous nucleic acid sequences comprising a single-stranded region capable of hybridization with the nucleic acid sequence to be detected adjacent to a single-stranded region not capable of such hybridization, said probe being covalently bound to at least one alkylating moiety, which is in turn bound to a divalent organic moiety having the formula: ##STR18## where Y is 0, 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 alkylating and label moieties.

Claim 19

A labeled single-stranded nucleic acid hybridization probe for detecting homologous nucleic acid sequences comprising a single-stranded region capable of hybridization with the nucleic acid sequence to be detected adjacent to a single-stranded region not capable of such hybridization, said probe being covalently bound to at least one alkylating moiety, which is in turn bound to a divalent organic moiety having the formula: ##STR18## where Y is 0, 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 alkylating and label moieties.

Claim 20

The probe of claim 15 wherein the alkylating moiety is a 4'-methylene-substituted-4,5',8-trimethylpsoralen moiety and 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 21

The probe of claim 16 wherein the bound conjugate of divalent organic moiety and monovalent label moiety has the formula: ##STR19## wherein Y is NH.

Claim 22

The probe of claim 15 wherein the nucleic acid is DNA.

Claim 23

The probe of claim 15 wherein the nucleic acid sequence to be detected is characteristic of a pathogenic microbe, is in a gene involved in HLA typing, is an amplified oncogene, or is associated with a genetic or infectious disease.

Claim 24

A complex comprising the probe of claim 19 and a polypeptide, lectin, or antibody capable of forming a complex with the label moiety of said probe.

Claim 25

The complex of claim 20 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 a reaction product.

Claim 26

A complex comprising the probe of claim 19 containing biotin as the label moiety and a conjugate of streptavidin and a peroxidase.

Claim 27

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, animal cell membranes, body fluids, viral capsids, bacterial cell walls, bacterial membranes, or tissues of the sample and to expose and separate the strands of the nucleic acid or acids in the sample; (b) contacting the exposed and separated strands with an effective amount of the hybridization probe of claim 15 under hybridization conditions; and (c) detecting whether hybridization of homologous nucleic acid sequences to the probe has occurred by means of the label moiety on the probe.

Claim 28

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, animal cell membranes, body fluids, viral capsids, bacterial cell walls, bacterial membranes, or tissues of the sample and to expose and separate the strands of the nucleic acid or acids in the sample; (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 15 under hybridization conditions; and (e) detecting hybridization of any homologous single-stranded nucleic acid sequences by means of the label moiety on the probe.

Claim 29

The process of claim 24 wherein the homologous sequence or sequences to be detected are characteristic of a pathogenic microbe or are associated with an infectious disease.

Claim 30

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 membrane; (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 membrane at about the same site on the membrane 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 membrane with the probe to be employed; (h) contacting said affixed single-stranded DNA sample with an effective amount of the hybridization probe of claim 17 containing a single-stranded HLA gene as the hybridizing region under hybridization conditions; (i) washing said sample of unhybridized probe; (j) detecting hybridization of any homologous single-stranded nucleic acid sequence or sequences present 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 31

The process of claim 26 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.

Claim 32

A marker mix comprising a mixture of two or more single-stranded nucleic acids of different lengths or specificities which are covalently bound to at least one alkylating moiety, which is in turn bound to a divalent organic moiety having the formula: ##STR20## where Y is 0, 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 alkylating and label moieties.

Claim 33

A marker mix comprising a mixture of two or more single-stranded nucleic acids of different lengths or specificities which are covalently bound to at least one alkylating moiety, which is in turn bound to a divalent organic moiety having the formula: ##STR20## where Y is 0, 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 alkylating and label moieties.