Methods for rapid screening of polymorphisms, mutations and methylation
First Claim
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1. A method of identifying at least two nucleic acid alterations in a single target nucleic acid, which comprises:
- (a) preparing the target nucleic acid and a control nucleic acid;
(b) hybridizing the target nucleic acid with the control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type nucleic acid corresponding to the target nucleic acid, and wherein a mismatch is formed between the control and the target nucleic acid at each location where a difference is present;
(c) removing any 5′
phosphate groups or 3′
hydroxyl groups from the heteroduplex nucleic acid ends by circularizing said heteroduplex nucleic acid and selectively degrading any non-circularized heteroduplex nucleic acids;
(d) cleaving said circularized heteroduplex nucleic acid at the site of any mismatch using any agent that generates a single or double strand break at the site of the mismatch, thereby creating free 5′
P or 3′
OH ends at the site of said any mismatch;
(e) ligating a linker at the 5′
phosphate group at any of the newly generated 5′
phosphate groups, or ligating a linker at any of the newly generated 3′
hydroxyl groups;
(f) selectively amplifying the cleaved, heteroduplex nucleic acid fragments which have a linker ligated thereto using a polymerase chain reaction; and
(g) determining if at least two different nucleic acid fragments are present to identify the at least two nucleic acid alterations present in the target nucleic acid.
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Abstract
The present method is directed to methods of detecting mismatches, polymorphisms, and methylation in multiple genes or the same gene in multiple individuals.
52 Citations
17 Claims
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1. A method of identifying at least two nucleic acid alterations in a single target nucleic acid, which comprises:
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(a) preparing the target nucleic acid and a control nucleic acid; (b) hybridizing the target nucleic acid with the control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type nucleic acid corresponding to the target nucleic acid, and wherein a mismatch is formed between the control and the target nucleic acid at each location where a difference is present; (c) removing any 5′
phosphate groups or 3′
hydroxyl groups from the heteroduplex nucleic acid ends by circularizing said heteroduplex nucleic acid and selectively degrading any non-circularized heteroduplex nucleic acids;(d) cleaving said circularized heteroduplex nucleic acid at the site of any mismatch using any agent that generates a single or double strand break at the site of the mismatch, thereby creating free 5′
P or 3′
OH ends at the site of said any mismatch;(e) ligating a linker at the 5′
phosphate group at any of the newly generated 5′
phosphate groups, or ligating a linker at any of the newly generated 3′
hydroxyl groups;(f) selectively amplifying the cleaved, heteroduplex nucleic acid fragments which have a linker ligated thereto using a polymerase chain reaction; and (g) determining if at least two different nucleic acid fragments are present to identify the at least two nucleic acid alterations present in the target nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of identifying at least two nucleic acid alterations in a single target nucleic acid, which comprises:
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(a) preparing a target nucleic acid that comprises a specific recognition sequence for a restriction endonuclease enzyme, wherein its wild-type allele lacks; (b) hybridizing the target nucleic acid with a control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type allele corresponding to the target nucleic acid and wherein a mismatch is formed between the wild type and the target nucleic acid whenever a difference is present; (c) removing any 5′
phosphate groups or 3′
hydroxyl groups from the heteroduplex nucleic acid ends by circularizing said heteroduplex nucleic acid and selectively degrading any non-circularized heteroduplex nucleic acids;(d) cleaving said circularized heteroduplex nucleic acid with a restriction endonuclease enzyme to create a double strand break at the site of any mismatch, generating free 5′
P or 3′
OH ends at the site of said any mismatch;(e) ligating a linker at any of the newly generated 5′
phosphate groups at the sites of cleavage when 5′
phosphate groups have been removed, or ligating a linker at any of the newly generated 3′
hydroxyl groups at the site of cleavage, when 3′
hydroxyl groups have been removed;(f) selectively amplifying the nucleic acid fragments amplified in step (e) to identify if at least two alterations are present, wherein the selective amplification of the nucleic acid fragments is performed via nested-PCR using primers internal to the nucleic acid fragments. - View Dependent Claims (14, 15, 16)
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17. A method of identifying at least two nucleic acid alterations in a single target nucleic acid, which comprises:
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(a) preparing the target nucleic acid and a control nucleic acid; (b) hybridizing the target nucleic acid with the control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type nucleic acid corresponding to the target nucleic acid and wherein a mismatch is formed between the wild type and the target nucleic acid whenever a difference is present; (c) removing any 5′
phosphate groups or 3′
hydroxyl groups from the heteroduplex nucleic acid ends;(d) cleaving the heteroduplex nucleic acid at the site of any mismatch using any combination of agents that generate blunt-ended double strand breaks at the site of mismatch thereby creating free 5′
P or 3′
OH ends at the site of any mismatch;(e) ligating a linker at any of the 5′
phosphate group at the newly generated 5′
phosphate groups at the site of cleavage when 5′
phosphate groups have been removed, or ligating a linker at any of the newly generated 3′
hydroxyl groups at the site of cleavage when 3′
hydroxyl groups have been removed;(f) selectively amplifying the nucleic acid fragments which have a linker ligated thereto using polymerase chain reaction; and (g) detecting if at least two different nucleic acid fragments are present to identify the at least two alterations in the target nucleic acid, wherein following cleavage of the heteroduplex nucleic acid at the site of mismatch, S1 nuclease is used to convert any single strand breaks to blunt-ended double strand breaks.
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Specification