Nonradiometric polynucleotide probes

Claim 2

A polynucleotide probe composition capable of forming hybrids with target polynucleotides which are detectable by means of an enzymatic reaction cascade of the formula ##STR9## wherein n is an integer from 2 to 500; A is a polynucleotide having regions substantially complementary to a target polynucleotide; and Z 1 through Z n , which are the same or different are nucleotide moieties which collectively form a polynucleotide sequence; provided that at least one of nucleotide moieties Z 1 through Z n comprises a moiety X, where X is a moiety comprising a proteolytic enzyme capable of enzymatically activating a zymogen to initiate a detectable enzymatic reaction cascade.

Claim 3

A composition according to claim 1, wherein A is a polynucleotide comprising a nucleotide sequence substantially complementary to all or part of a gene associated with an antibiotic resistance phenotype capable of expression by a pathogen.

Claim 4

A composition according to claim 1, wherein A is a polynucleotide comprising a nucleotide sequence substantially complementary to all or part of a mammalian DNA associated with a genetic disorder.

Claim 5

A diagnostic kit for detecting the presence of a target polynucleotide characteristic of or associated with a pathogen, a pathogen capable of expressing an antibiotic resistance phenotype, or a genetic disorder in mammals, said kit comprising a composition according to claim 1, and a detection substrate comprising a zymogen which, upon activation by a zymogen activator, is capable of initiating a detectable enzymatic reaction cascade.

Claim 6

A composition according to claim 1 wherein A is a polynucleotide comprising a nucleotide sequence substantially complementary to all or part of a structural gene characteristic of a pathogen.

Claim 7

A composition according to claim 5, wherein the pathogen is a bacillus.

Claim 8

A composition according to claim 5, wherein the pathogen is a mycobacterium, actinomycete, or spirochete.

Claim 9

A composition according to claim 5, wherein the pathogen is a rickettsia, chlamydia, or mycoplasma.

Claim 10

A composition according to claim 5, wherein the pathogen is a fungus.

Claim 11

A composition according to claim 5, wherein the pathogen is a virus.

Claim 12

A composition according to claim 5, wherein the pathogen is a eukaryote.

Claim 13

A composition according to claim 11, wherein the pathogen is a multicellular organism.

Claim 14

A method for detecting the presence of a target polynucleotide in a sample, comprising: (a) contacting, under hybridizing conditions, a polynucleotide probe composition of claim 1 with the sample, to permit formation of probe/target hybrids; (b) separating the probe/target hybrids from unhybridized probe composition; and (c) contacting the probe/target hybrids with a zymogen to initiate a detectable enzymatic reaction cascade.

Claim 15

A method according to claim 13, wherein the target polynucleotide is associated with an antibiotic resistance phenotype capable of expression by a pathogen.

Claim 16

A method according to claim 13, wherein the target polynucleotide represents all or part of a mammalian DNA associated with a genetic disorder.

Claim 17

A method according to claim 13, wherein the target polynucleotide is characteristic of a pathogen.

Claim 18

A method according to claim 16, wherein the pathogen is a bacillus.

Claim 19

A method according to claim 16, wherein the pathogen is a mycobacterium, actinomycete, spirochete, rickettsia, chlamydia, mycoplasma, or fungus.

Claim 20

A method according to claim 16, wherein the pathogen is virus.

Claim 21

A method according to claim 16, wherein the pathogen is a eukaryote.

Claim 22

A polynucleotide probe composition according to claim 1, said composition having the formula ##STR10## wherein n is an integer from 2 to 500; A is a polynucleotide having regions substantially complementary to a target polynucleotide; and Z 1 through Z n , which are the same or different, are nucleotide moieties which collectively form a polynucleotide sequence; said nucleotide moieties having the formula ##STR11## wherein R 1 is BR 5 ; where B is a base residue; and R 5 is H or X, where X is moiety comprising a proteolytic enzyme capableof enzymatically activating a zymogen to initiate a detectable enzymatic reaction cascade; R 2 and R 3 are H, OH, X, a phosphate group or groups, an adjacent nucleotide moiety, a phosphate group covalently linked to a moiety X or to an adjacent nucleotide moiety, or a phosphate group covalently linked to a moiety X and an adjacent nucleotide moiety; and R 4 is H, OH, a phosphate group, or X; provided that, for at least one of nucleotide moieties Z 1 through Z n , R 1 is BR 5 and R 5 is X; or, R 2 , R 3 , or R 4 comprises a moiety X.

Claim 23

A composition according to claim 21, wherein for at least one of nucleotide moieties Z 1 through Z n , R 2 comprises a moiety X.

Claim 24

A composition according to claim 21, wherein for at least one of nucleotide moieties Z 1 through Z n , R 3 comprises a moiety X.

Claim 25

A composition according to claim 21, wherein for at least one of nucleotide moieties Z 1 through Z n , R 4 is X.

Claim 26

A composition according to claim 21, wherein for at least one of nucleotide moieties Z 1 through Z n , ##STR12## where R 5 is X.

Claim 27

A composition according to claim 25, wherein X is LE, where L is a linking group; and E is an enzyme having zymogen-activating activity.

Claim 28

A composition according to claim 26 wherein E is enteropeptidase.

Claim 29

A composition according to claim 26, wherein E is a plasminogen activator.

Claim 30

A composition according to claim 28, wherein E is urokinase.

Claim 31

A composition according to claim 21, wherein for at least one of nucleotide moieties Z 1 through Z n , ##STR13## where R 5 is X.

Claim 32

A composition according to claim 30, wherein X is LE, where L is a linking group; and E is an enzyme having zymogen-activating activity.

Claim 33

A composition according to claim 31, wherein E is enteropeptidase.

Claim 34

A composition according to claim 31 wherein E is a plasminogen activator.

Claim 35

A composition according to claim 31 wherein E is urokinase.

Claim 36

A composition according to claim 21, wherein for at least one of nucleotide moieties Z 1 through Z n , ##STR14## where R 5 is X.

Claim 37

A composition according to claim 35, wherein X is LE, where L is a linking group; and E is an enzyme having proteolytic activity.

Claim 38

A composition according to claim 36, wherein E is enteropeptidase.

Claim 39

A composition according to claim 36, wherein E is a plasminogen activator.

Claim 40

A composition according to claim 38, wherein E is urokinase.

Claim 41

A composition according to claim 21, wherein for at least one of nucleotide moieties Z 1 through Z n , ##STR15## where R 5 is X.

Claim 42

A composition according to claim 40, wherein X is LE, where L is a linking group; and E is an enzyme having zymogen-activating activity.

Claim 43

A composition according to claim 41, wherein E is enteropeptidase.

Claim 44

A composition according to claim 41, wherein E is a plasminogen activator.

Claim 45

A composition according to claim 43, wherein E is urokinase.