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 in a plurality of differing target nucleotide sequences comprising:
- providing a sample potentially containing target nucleotide sequences differing by one or more single-base changes, insertions, deletions, or translocations;
providing oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe comprising a target-specific portion and (b) a second oligonucleotide probe comprising a target-specific portion and a further portion, wherein the further portion comprises a nucleic acid sequence of greater than 16 nucleotides that differs for each different target-specific portion, wherein the further portions of each set hybridize to their complementary portions under uniform hybridization conditions, wherein the nucleic acid sequence of a complement to one further portion differs from the nucleic acid sequence of a complement to another further portion, when aligned to each other, by at least 25% to minimize cross-hybridization of one further portion to a complementary portion of another further portion;
providing a ligase;
blending the sample, the oligonucleotide probe sets, and the ligase to form a ligation detection reaction mixture;
subjecting the ligation detection reaction mixture to one or more ligation detection reaction cycles to form a ligated product sequence comprising (a) the target-specific portions and (b) the further portion if its target nucleotide sequence is present in the sample, wherein the further portion is comprised of a nucleotide sequence which is distinct from that of the target-specific portions to prevent hybridization between the target specific portions and the further portion; and
detecting and distinguishing the presence of different target nucleotide sequences in the sample based on the further portion of the ligated product sequence.
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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.
147 Citations
28 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 in a plurality of differing target nucleotide sequences 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 oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe comprising a target-specific portion and (b) a second oligonucleotide probe comprising a target-specific portion and a further portion, wherein the further portion comprises a nucleic acid sequence of greater than 16 nucleotides that differs for each different target-specific portion, wherein the further portions of each set hybridize to their complementary portions under uniform hybridization conditions, wherein the nucleic acid sequence of a complement to one further portion differs from the nucleic acid sequence of a complement to another further portion, when aligned to each other, by at least 25% to minimize cross-hybridization of one further portion to a complementary portion of another further portion; providing a ligase; blending the sample, the oligonucleotide probe sets, and the ligase to form a ligation detection reaction mixture; subjecting the ligation detection reaction mixture to one or more ligation detection reaction cycles to form a ligated product sequence comprising (a) the target-specific portions and (b) the further portion if its target nucleotide sequence is present in the sample, wherein the further portion is comprised of a nucleotide sequence which is distinct from that of the target-specific portions to prevent hybridization between the target specific portions and the further portion; and detecting and distinguishing the presence of different target nucleotide sequences in the sample based on the further portion of the ligated product sequence. - 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)
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Specification