US5834181A
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US
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High throughput screening method for sequences or genetic alterations in nucleic acids
Assignee: Esoterix Genetic Laboratories LLC
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Filing: 1996-09-13
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Granted: 1998-11-10
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Expires: 2016-09-13
Abstract
Abstract The present invention provides methods for identifying genetic alterations in a target sequence present in a nucleic acid sample, comprising immobilizing samples on a support, contacting the samples simultaneously with different purine and pyrimidine containing polymers under conditions of hybridization, separating the hybridized polymers from the samples; and identifying the hybridized polymers to identify the genetic alteration(s). The present invention also provides methods for identifying target sequences present in a nucleic acid sample, comprising immobilizing nucleic acid samples on a support, contacting the samples simultaneously different purine and pyrimidine containing polymers under hybridization conditions, separating the polymers from the complementary target sequence(s), and identifying the hybridized polymers to identify the target sequence(s). Further provided by the present invention are methods for identifying randomly permuted genetic alterations in a target sequence present in a nucleic acid sample, comprising immobilizing nucleic acid samples on a support, contacting the samples simultaneously with different purine and pyrimidine containing polymers under hybridization conditions, detecting hybridization between the polymers and the complementary target sequences, to identify the target sequence(s), separating the hybridized polymers from the complementary target sequences; and identifying randomly permuted genetic alterations present in the target sequence.
Inventors
- 👤 Anthony P. Shuber
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 22:15
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6834
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
107 words
A method for identifying one or more genetic alterations in a target sequence present in a nucleic acid sample, comprising the steps of: (i) immobilizing a plurality of nucleic acid samples on a support; (ii) contacting said immobilizes samples simultaneously with a multiplicity of different purine and pyrimidine containing polymers under conditions wherein individual purine and pyrimidine containing polymers hybridize to a complementary target sequence in said immobilized samples; (iii) separating the hybridized purine and pyrimidine containing polymers from said immobilized samples; and (iv) identifying the hybridized purine and pyrimidine containing polymers, wherein the identification of the hybridized purine and pyrimidine containing polymers identifies said genetic alteration(s).
Claim 2
Independent Claim
107 words
A method for identifying one or more genetic alterations in a target sequence present in a nucleic acid sample, comprising the steps of: (i) immobilizing a plurality of nucleic acid samples on a support; (ii) contacting said immobilizes samples simultaneously with a multiplicity of different purine and pyrimidine containing polymers under conditions wherein individual purine and pyrimidine containing polymers hybridize to a complementary target sequence in said immobilized samples; (iii) separating the hybridized purine and pyrimidine containing polymers from said immobilized samples; and (iv) identifying the hybridized purine and pyrimidine containing polymers, wherein the identification of the hybridized purine and pyrimidine containing polymers identifies said genetic alteration(s).
Claim 3
Independent Claim
104 words
A method for identifying one or more target sequences present in a nucleic acid sample, comprising the steps of: (i) immobilizing a plurality of nucleic acid samples on a support; (ii) contacting said immobilized samples simultaneously with a multiplicity of different purine and pyrimidine containing polymers under conditions wherein individual purine and pyrimidine containing polymers hybridize to a complementary target sequence in said immobilized samples; (iii) separating the hybridized purine and pyrimidine containing polymers from said complementary target sequence(s); and (iv) identifying the hybridized purine and pyrimidine containing polymers, wherein the identification of the hybridized purine and pyrimidine containing polymers identifies said target sequence(s).
Claim 4
Independent Claim
104 words
A method for identifying one or more target sequences present in a nucleic acid sample, comprising the steps of: (i) immobilizing a plurality of nucleic acid samples on a support; (ii) contacting said immobilized samples simultaneously with a multiplicity of different purine and pyrimidine containing polymers under conditions wherein individual purine and pyrimidine containing polymers hybridize to a complementary target sequence in said immobilized samples; (iii) separating the hybridized purine and pyrimidine containing polymers from said complementary target sequence(s); and (iv) identifying the hybridized purine and pyrimidine containing polymers, wherein the identification of the hybridized purine and pyrimidine containing polymers identifies said target sequence(s).
Claim 5
Dependent Claim
19 words
The method of claim 2, wherein the nucleic acid sample is suspected of containing more than one target sequences.
Claim 6
Dependent Claim
24 words
The method of claim 2, wherein the target sequence is selected from the group consisting of viral, bacterial, fungal, and protozoal nucleic acid sequences.
Claim 7
Independent Claim
130 words
A method for identifying one or more randomly permuted genetic alterations in a target sequence present in a nucleic acid sample, comprising the steps of: (i) immobilizing a plurality of nucleic acid samples on a support; (ii) contacting said immobilized samples simultaneously with a multiplicity of different purine and pyrimidine containing polymers under conditions wherein individual purine and pyrimidine containing polymers hybridize to a complementary target sequence in said immobilized samples; (iii) detecting hybridization between said purine and pyrimidine containing polymers and said complementary target sequences, wherein the detection of the hybridized purine and pyrimidine containing polymers identifies said target sequence(s); (iv) separating said hybridized purine and pyrimidine containing polymers from said complementary target sequences; and (v) identifying one or more randomly permuted genetic alterations present in said target sequence.
Claim 8
Independent Claim
130 words
A method for identifying one or more randomly permuted genetic alterations in a target sequence present in a nucleic acid sample, comprising the steps of: (i) immobilizing a plurality of nucleic acid samples on a support; (ii) contacting said immobilized samples simultaneously with a multiplicity of different purine and pyrimidine containing polymers under conditions wherein individual purine and pyrimidine containing polymers hybridize to a complementary target sequence in said immobilized samples; (iii) detecting hybridization between said purine and pyrimidine containing polymers and said complementary target sequences, wherein the detection of the hybridized purine and pyrimidine containing polymers identifies said target sequence(s); (iv) separating said hybridized purine and pyrimidine containing polymers from said complementary target sequences; and (v) identifying one or more randomly permuted genetic alterations present in said target sequence.
Claim 9
Dependent Claim
15 words
The method of claim 1, wherein said genetic alterations comprise nucleotide insertions, deletions, or substitutions.
Claim 10
Dependent Claim
19 words
The method of claim 1, wherein the nucleic acid samples are suspected of containing one or more genetic alterations.
Claim 11
Dependent Claim
31 words
The method of claim 7, wherein the genetic alteration is associated with a disease selected from the group consisting of cystic fibrosis, β-thalassemia, Tay-Sachs disease, sickle cell anemia, and Gaucher's disease.
Claim 12
Dependent Claim
20 words
The method of claim 1, wherein said purine and pyrimidine containing polymers are from 16 to 25 bp in length.
Claim 13
Dependent Claim
21 words
The method of claim 1, wherein the target sequence is amplified from the nucleic acid samples prior to the immobilizing step.
Claim 14
Dependent Claim
19 words
The method of claim 1, wherein the amplified sequence is from 80 bp to about 30 kbp in length.
Claim 15
Dependent Claim
54 words
The method of claim 1, wherein said purine and pyrimidine containing polymers are of approximately the same length and said contacting step occurs in the presence of an effective concentration of an agent that eliminates disparities in the melting temperatures of hybrids formed between said purine and pyrimidine containing polymers and said target sequence(s).
Claim 16
Dependent Claim
13 words
The method of claim 12, wherein the agent is a quaternary ammonium salt.
Claim 17
Dependent Claim
74 words
The method of claim 1, wherein the identifying step comprises: (a) contacting said hybridized purine and pyrimidine containing polymers with a multiplicity of complementary oligonucleotides comprising (i) sequences complementary to said polymers and (ii) additional predetermined colinear sequences; (b) performing enzymatic sequencing, wherein said polymers serve as primers and the complementary oligonucleotides serve as templates for enzymatic sequencing; and (c) identifying the predetermined colinear sequences as an indicator of the presence of said polymers.
Claim 18
Dependent Claim
88 words
The method of claim 1, wherein the identifying step comprises: (a) contacting said hybridized purine and pyrimidine containing polymers with a multiplicity of complementary oligonucleotides comprising (i) sequences complementary to said polymers and (ii) additional predetermined colinear sequences; (b) performing a single extension reaction, wherein said polymers serve as primers and the complementary oligonucleotides serve as templates for the extension reaction; (c) performing enzymatic sequencing of the products of the extension reaction; and (d) identifying the predetermined colinear sequences as an indicator of the presence of said polymers.
Claim 19
Dependent Claim
25 words
The method of claim 1, wherein said purine and pyrimidine polymers are each provided with a molecular weight modifying entity having a unique molecular weight.
Claim 20
Dependent Claim
17 words
The method of claim 1, wherein said purine and pyrimidine polymers are provided with a detectable label.
Claim 21
Dependent Claim
27 words
The method of claim 1 wherein the purine and pyrimidine containing polymers that do not hybridize to said immobilized samples are removed prior to said identifying step.
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