Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
First Claim
1. A method for identifying a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations, said method comprising:
- providing a sample potentially containing target nucleotide sequences differing by one or more single-base changes, insertions, deletions, or translocations;
providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label;
providing a ligase;
blending the sample, the plurality of oligonucleotide probe sets, and the ligase to form a mixture;
subjecting the mixture to one or more ligase detection reaction cycles to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions, and (c) the detectable reporter label, if their respective target nucleotide is present in the sample;
providing a solid support with capture oligonucleotides immobilized at particular sites, wherein the capture oligonucleotides, each having greater than 16 nucleotides, have nucleotide sequences complementary to the addressable array-specific portions with the capture oligonucleotides hybridizing to their complementary addressable array specific portion under uniform hybridization conditions, wherein adjacent capture oligonucleotides on the solid support which are complementary to the addressable array-specific portions corresponding to single-base changes, insertions, deletions, or translocations at different locations in the target nucleotide sequences differ in their nucleotide sequences, when aligned to each other, by at least 25% of the nucleotides to minimize cross hybridization between non-complementary capture oligonucleotides and addressable array-specific portions, and, wherein the solid support and the capture oligonucleotides form an addressable array;
contacting the mixture, after said subjecting, with the solid support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the solid support at the site with the complementary capture oligonucleotide; and
detecting and distinguishing the reporter labels of ligated product sequences captured to the solid support at particular sites, thereby indicating the presence of target nucleotide sequences in the sample, wherein the oligonucleotide probe sets are configured so that the addressable array-specific portion is comprised of a nucleotide sequence which is distinct from that of the target-specific portions, in order to prevent hybridization between the target-specific portions and the capture oligonucleotides as well as between the target nucleotide sequence and the addressable array-specific portion.
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Abstract
The present invention describes a method for identifying one or more of a plurality of sequences differing by one or more single base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences. The method includes a ligation phase, a capture phase, and a detection phase. The ligation phase utilizes a ligation detection reaction between one oligonucleotide probe, which has a target sequence-specific portion and an addressable array-specific portion, and a second oligonucleotide probe, having a target sequence-specific portion and a detectable label. After the ligation phase, the capture phase is carried out by hybridizing the ligated oligonucleotide probes to a solid support with an array of immobilized capture oligonucleotides at least some of which are complementary to the addressable array-specific portion. Following completion of the capture phase, a detection phase is carried out to detect the labels of ligated oligonucleotide probes hybridized to the solid support.
157 Citations
51 Claims
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1. A method for identifying a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations, said method comprising:
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providing a sample potentially containing target nucleotide sequences differing by one or more single-base changes, insertions, deletions, or translocations; providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and an addressable array-specific portion and (b) a second oligonucleotide probe, having a target-specific portion and a detectable reporter label; providing a ligase; blending the sample, the plurality of oligonucleotide probe sets, and the ligase to form a mixture; subjecting the mixture to one or more ligase detection reaction cycles to form a ligated product sequence containing (a) the addressable array-specific portion, (b) the target-specific portions, and (c) the detectable reporter label, if their respective target nucleotide is present in the sample; providing a solid support with capture oligonucleotides immobilized at particular sites, wherein the capture oligonucleotides, each having greater than 16 nucleotides, have nucleotide sequences complementary to the addressable array-specific portions with the capture oligonucleotides hybridizing to their complementary addressable array specific portion under uniform hybridization conditions, wherein adjacent capture oligonucleotides on the solid support which are complementary to the addressable array-specific portions corresponding to single-base changes, insertions, deletions, or translocations at different locations in the target nucleotide sequences differ in their nucleotide sequences, when aligned to each other, by at least 25% of the nucleotides to minimize cross hybridization between non-complementary capture oligonucleotides and addressable array-specific portions, and, wherein the solid support and the capture oligonucleotides form an addressable array; contacting the mixture, after said subjecting, with the solid support under conditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the solid support at the site with the complementary capture oligonucleotide; and detecting and distinguishing the reporter labels of ligated product sequences captured to the solid support at particular sites, thereby indicating the presence of target nucleotide sequences in the sample, wherein the oligonucleotide probe sets are configured so that the addressable array-specific portion is comprised of a nucleotide sequence which is distinct from that of the target-specific portions, in order to prevent hybridization between the target-specific portions and the capture oligonucleotides as well as between the target nucleotide sequence and the addressable array-specific portion. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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51. A method for identifying a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising:
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producing ligation products from a reaction mixture, wherein said reaction mixture comprises; a ligase; target nucleotide sequences; and oligonucleotide probe sets, each probe set including (a) a first oligonucleotide probe comprising a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence and (b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence, wherein a ligation product comprising the first and the second target-specific portions is produced when the first and the second target-specific portions are hybridized to said corresponding target nucleotide sequence, but is not produced when the first and the second target-specific portions are hybridized with one or more mismatches to a nucleotide sequence present in said reaction mixture, wherein each of said ligation products comprises a ligated sequence which includes (1) the first target-specific portion of the first oligonucleotide probe in a corresponding probe set, (2) the second target-specific portion of the second oligonucleotide probe in said corresponding probe set, and (3) an addressable array-specific portion, or complement thereof, that is comprised of a nucleotide sequence that is distinct from the target-specific portions, in order to prevent hybridization between the target-specific portions and the capture oligonucleotides as well as between the target nucleotide sequence and the addressable array-specific portion; capturing polynucleotides comprising said ligated sequences to a solid support, wherein each polynucleotide further comprises a reporter label, and the reporter label together with the addressable array-specific portion in that polynucleotide distinguish the ligated sequence in that polynucleotide from those in other polynucleotides with the capture oligonucleotides, each having greater than 16 nucleotides, hybridizing to their complementary addressable array specific portion under uniform hybridization conditions, wherein adjacent capture oligonucleotides on the solid support which are complementary to the addressable array-specific portions corresponding to single-base changes, insertions, deletions, or translocations at different locations in the target nucleotide sequences differ in their nucleotide sequences, when aligned to each other, by at least 25% of the nucleotides to minimize cross hybridization between non-complementary capture oligonucleotides and addressable array-specific portions; and detecting the reporter labels and the identities of the addressable sequences in said captured polynucleotides to indicate the presence of one or more target nucleotide sequences in said reaction mixture.
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Specification