US4883750A — Detection of specific sequences in nucleic acids

Claim 1

Independent Claim 222 words

A method of testing for the presence or absence of a target sequence in a sample containing DNA, the steps comprising: (a) denaturing the sample DNA; (b) treating the denatured DNA with a first probe which is complementary to a first portion of the target sequence to produce a duplex of first probe and denatured DNA; (c) treating the duplex formed in step (b) with a second probe, which is shorter than the first probe and ligatable therewith, and which is complementary to the remaining portion of the target sequence; (d) providing conditions for hybridization of the first probe and second probe to the target sequence in steps (b) and (c) wherein, by controlling stringency, the first probe does not bind substantially to the denature DNA which does not contain the first portion and wherein the second probe does not bind substantially to the denatured DNA which does not contain the remaining portion, the first probe and the second probe being of sufficient length that they can be stably hybridized to the target sequence long enough to be ligated to each other, and wherein at least one of the first and second probe is labeled; (e) ligating first and second probe which are hybridized to the target sequence; (f) removing unligated probe after step (e); (g) testing for the presence of label.

Claim 2

Independent Claim 222 words

A method of testing for the presence or absence of a target sequence in a sample containing DNA, the steps comprising: (a) denaturing the sample DNA; (b) treating the denatured DNA with a first probe which is complementary to a first portion of the target sequence to produce a duplex of first probe and denatured DNA; (c) treating the duplex formed in step (b) with a second probe, which is shorter than the first probe and ligatable therewith, and which is complementary to the remaining portion of the target sequence; (d) providing conditions for hybridization of the first probe and second probe to the target sequence in steps (b) and (c) wherein, by controlling stringency, the first probe does not bind substantially to the denature DNA which does not contain the first portion and wherein the second probe does not bind substantially to the denatured DNA which does not contain the remaining portion, the first probe and the second probe being of sufficient length that they can be stably hybridized to the target sequence long enough to be ligated to each other, and wherein at least one of the first and second probe is labeled; (e) ligating first and second probe which are hybridized to the target sequence; (f) removing unligated probe after step (e); (g) testing for the presence of label.

Claim 3

Independent Claim 226 words

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, which method comprises: (a) treating the denatured nucleic acid with a diagnostic probe complementary to a diagnostic portion of the target sequence, and with a contiguous probe complementary to a nucleotide sequence in the target sequence which is contiguous with the diagnostic portion, under conditions of stringency wherein the diagnostic probe and the contiguous probe would become hybridized to the denatured nucleic acid if the target sequence were present in the sample, said conditions of stringency such that the diagnostic probe remains bound substantially only to sample nucleic acid containing the target sequence, the diagnostic probe and the contiguous probe being of sufficient length to be able to stably hybridize to the target sequence long enough so that the diagnostic probe and the contiguous probe can be covalently attached to each other, and wherein at least one of the diagnostic and contiguous probes is labeled; (b) covalently attaching the diagnostic probe to the contiguous probe after the sample has been treated with the diagnostic probe and the contiguous probe in step (a) to produce a target probe complementary to the target sequence; (c) removing the probes not attached in step (b); and (d) testing for the presence of label in the sample after step (c).

Claim 4

Independent Claim 226 words

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, which method comprises: (a) treating the denatured nucleic acid with a diagnostic probe complementary to a diagnostic portion of the target sequence, and with a contiguous probe complementary to a nucleotide sequence in the target sequence which is contiguous with the diagnostic portion, under conditions of stringency wherein the diagnostic probe and the contiguous probe would become hybridized to the denatured nucleic acid if the target sequence were present in the sample, said conditions of stringency such that the diagnostic probe remains bound substantially only to sample nucleic acid containing the target sequence, the diagnostic probe and the contiguous probe being of sufficient length to be able to stably hybridize to the target sequence long enough so that the diagnostic probe and the contiguous probe can be covalently attached to each other, and wherein at least one of the diagnostic and contiguous probes is labeled; (b) covalently attaching the diagnostic probe to the contiguous probe after the sample has been treated with the diagnostic probe and the contiguous probe in step (a) to produce a target probe complementary to the target sequence; (c) removing the probes not attached in step (b); and (d) testing for the presence of label in the sample after step (c).

Claim 5

Dependent Claim 10 words

The method of claim 2 wherein the label is radioactive.

Claim 6

Dependent Claim 29 words

The method of claim 3 wherein the label is 32 P at the 5' terminus of one probe to be ligated to the 3' terminus of the other probe.

Claim 7

Dependent Claim 20 words

The method of claim 4 wherein step (d) of testing comprises measuring the amount of radioactivity present in the sample.

Claim 8

Dependent Claim 31 words

The method of claim 2 wherein step (d) of testing includes removing the target probe formed in step (b) and retaining any target probe so removed to provide a second sample.

Claim 9

Dependent Claim 20 words

The method of claim 6 wherein in the step of removing the target probe is eluted by increasing the stringency.

Claim 10

Dependent Claim 21 words

The method of claim 6 wherein in the step of removing, the target probe is eluted by using a temperature gradient.

Claim 11

Dependent Claim 23 words

The method of claim 2 wherein before step (d) a hook is attached to the unlabeled one of the diagnostic and contiguous probes.

Claim 12

Dependent Claim 18 words

The method of claim 9 wherein the target probe is removed in step (d) by catching the hook.

Claim 13

Dependent Claim 12 words

The method of claim 10 wherein the hook comprises a biotin moiety.

Claim 14

Dependent Claim 18 words

The method of claim 11 wherein the hook is caught by using streptavidin to bind the biotin moiety.

Claim 15

Dependent Claim 16 words

The method of claim 6 wherein the second sample is tested for the presence of label.

Claim 16

Dependent Claim 10 words

The method of claim 13 wherein the label is radioactive.

Claim 17

Dependent Claim 29 words

The method of claim 14 wherein the label is 32 P at the 5' terminus of one probe to be ligated to the 3' terminus of the other probe.

Claim 18

Dependent Claim 21 words

The method of claim 15 wherein step (d) of testing comprises measuring the amount of radioactivity present in the second sample.

Claim 19

Independent Claim 266 words

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid using a diagnostic probe which is complementary to a diagnostic portion of the target sequence and a contiguous probe which is complementary to a portion of said target sequence which is contiguous with said diagnostic portion, wherein the diagnostic probe and the contiguous probe are of sufficient length that they can be stably hybridized to the target sequence long enough to be covalently attached to each other, and wherein one of the diagnostic probe and the contiguous probe will remain hybridized to the target sequence under a higher stringency than will the other, comprising the steps of: (a) hybridizing the one of the diagnostic and contiguous probes which will remain bound to its complementary portion of the target sequence under the higher stringency, said one of the diagnostic and contiguous probes hereinafter called the HS probe; (b) after Step (a) hybridizing the other of said diagnostic and contiguous probes which will remain bound to its complementary portion in the target sequence under conditions of lower stringency, said other of said diagnostic and contiguous probes hereinafter called the LS probe; (c) removing a substantial portion of said diagnostic probe which is not perfectly bound; (d) covalently attaching said LS probe and said HS probe where they are bound adjacent to each other to form a target probe complementary to said target sequence; (e) removing probe not attached in step (d); and (f) testing for the presence of target probe in the sample remaining after Step (e).

Claim 20

Independent Claim 266 words

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid using a diagnostic probe which is complementary to a diagnostic portion of the target sequence and a contiguous probe which is complementary to a portion of said target sequence which is contiguous with said diagnostic portion, wherein the diagnostic probe and the contiguous probe are of sufficient length that they can be stably hybridized to the target sequence long enough to be covalently attached to each other, and wherein one of the diagnostic probe and the contiguous probe will remain hybridized to the target sequence under a higher stringency than will the other, comprising the steps of: (a) hybridizing the one of the diagnostic and contiguous probes which will remain bound to its complementary portion of the target sequence under the higher stringency, said one of the diagnostic and contiguous probes hereinafter called the HS probe; (b) after Step (a) hybridizing the other of said diagnostic and contiguous probes which will remain bound to its complementary portion in the target sequence under conditions of lower stringency, said other of said diagnostic and contiguous probes hereinafter called the LS probe; (c) removing a substantial portion of said diagnostic probe which is not perfectly bound; (d) covalently attaching said LS probe and said HS probe where they are bound adjacent to each other to form a target probe complementary to said target sequence; (e) removing probe not attached in step (d); and (f) testing for the presence of target probe in the sample remaining after Step (e).

Claim 21

Dependent Claim 27 words

A method as in claim 17 wherein said diagnostic probe and said contiguous probe are ligatable to each other and wherein Step (d) is conducted using ligase.

Claim 22

Dependent Claim 18 words

A method as in claim 17 further comprising the step of: (g) measuring the amount of target probe.

Claim 23

Dependent Claim 24 words

A method as in claim 19 wherein one of said diagnostic and contiguous probes contains a label and Step (f) includes detecting said label.

Claim 24

Dependent Claim 18 words

A method as in claim 20 wherein Step (f) comprises increasing the stringency to remove the target probe.

Claim 25

Dependent Claim 22 words

A method as in claim 21 wherein Step (f) further comprises measuring the amount of label removed in removing the target probe.

Claim 26

Dependent Claim 27 words

A method as in claim 18 wherein Step (d) comprises covalently attaching the 5' end of said diagnostic probe with the 3' end of said contiguous probe.

Claim 27

Dependent Claim 27 words

A method as in claim 23 wherein said diagnostic probe and said contiguous probe are ligatable to each other and wherein Step (d) is conducted using ligase.

Claim 28

Dependent Claim 27 words

A method as in claim 17 wherein Step (d) comprises covalently attaching the 5' end of the contiguous probe with the 3' end of the diagnostic probe.

Claim 29

Dependent Claim 49 words

A method as in claim 19 wherein said target sequence corresponds to a genetic mutation from a known normal sequence, said method further comprising the step of comparing said amount of target probe from Step (g) with a calibrated standard for nucleic acid known to have said genetic mutation.

Claim 30

Independent Claim 233 words

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, the steps comprising: (a) treating the denatured nucleic acid with a diagnostic probe complementary to a diagnostic portion of the target sequence, and with a contiguous probe complementary to a nucleotide sequence in the target sequence which is contiguous with the diagnostic portion, under conditions wherein the diagnostic probe and the contiguous probe would become hybridized to the denatured nucleic acid if the target sequence were present in the sample, wherein at least one of the diagnostic and contiguous probes is labeled, wherein the diagnostic and contiguous probes can be covalently attached, wherein the diagnostic probe and the contiguous probe are of sufficient length to be stably hybridized to the target sequence long enough to be covalently attached to each other while they are hybridized to the target sequence, and wherein conditions of stringency are controlled so that said diagnostic probe is specifically bound; (b) after step (a), treating the sample to covalently attach the diagnostic probe to the contiguous probe where they are adjacent on the target sequence, to produce a target probe complementary to the target sequence; (c) removing the target probe formed in step (b) from the sample to form a second sample containing the target probe; and (d) testing for the presence of label in the second sample.

Claim 31

Independent Claim 233 words

A method of testing for the presence or absence of a target nucleotide sequence in a sample containing denatured nucleic acid, the steps comprising: (a) treating the denatured nucleic acid with a diagnostic probe complementary to a diagnostic portion of the target sequence, and with a contiguous probe complementary to a nucleotide sequence in the target sequence which is contiguous with the diagnostic portion, under conditions wherein the diagnostic probe and the contiguous probe would become hybridized to the denatured nucleic acid if the target sequence were present in the sample, wherein at least one of the diagnostic and contiguous probes is labeled, wherein the diagnostic and contiguous probes can be covalently attached, wherein the diagnostic probe and the contiguous probe are of sufficient length to be stably hybridized to the target sequence long enough to be covalently attached to each other while they are hybridized to the target sequence, and wherein conditions of stringency are controlled so that said diagnostic probe is specifically bound; (b) after step (a), treating the sample to covalently attach the diagnostic probe to the contiguous probe where they are adjacent on the target sequence, to produce a target probe complementary to the target sequence; (c) removing the target probe formed in step (b) from the sample to form a second sample containing the target probe; and (d) testing for the presence of label in the second sample.

Claim 32

Dependent Claim 20 words

The method of claim 27 wherein a hook is attached to the unlabeled one of the diagnostic and contiguous probes.

Claim 33

Dependent Claim 15 words

The method of claim 28 wherein the target probe is removed by catching the hook.

Claim 34

Dependent Claim 12 words

The method of claim 29 wherein the hook comprises a biotin moiety.

Claim 35

Dependent Claim 18 words

The method of claim 30 wherein the hook is caught by using streptavidin to bind the biotin moiety.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1992-11-27	1993-05-29	1993-11-28	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1996-11-27	1997-05-29	1997-11-28	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2000-11-27	2001-05-29	2001-11-28	$7700.00	Window Closed / Lapsed

Detection of specific sequences in nucleic acids

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