Method for detecting hybridization events in nucleic acids
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Abstract
The invention relates to materials and methods for detecting the presence of and/or quantifying a target polynucleotide in a sample by using probe polynucleotides attached to the surface of an array, which probes are at least partially complementary to the target polynucleotide. A kit for carrying out the invention is also provided.
13 Citations
14 Claims
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1-7. -7. (canceled)
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8. A method for detecting the presence of and/or quantifying a target polynucleotide in a sample comprising:
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(a) contacting a sample containing a target polynucleotide under hybridizing conditions with an array comprising at least one probe attached to a surface of the array, the probe comprising;
(i) a sequence segment (β
) having a 3′
end and a 5′
end, which sequence segment is complementary to at least a portion of the target polynucleotide,(ii) a first sequence segment (α
) covalently attached to one of the two ends of the sequence segment (β
),(iii) a second sequence segment (α
′
), which is at least partially complementary to the first sequence segment (′
), and is covalently attached to the other end of the sequence segment (β
),(iv) a means for attaching the probe to a surface of the array, the means being covalently linked to a free end of one of the first or second sequence segments (α
) or (α
′
),(v) a label attached to the end of the first or second sequence segments (α
) or (α
′
), which is not linked to the means for attaching the probe,the first and second sequence segments (α
) and (α
′
), respectively forming a duplex cleavage module containing a recognition site for a restriction endonuclease when the sequence segment (β
) does not hybridize with the target polynucleotide;
(b) treating the array with a restriction endonuclease, which cleaves at the recognition site formed when an (α
)-(α
′
) duplex cleavage module is formed;
(c) detecting and/or quantifying the presence of the target polynucleotide in the sample by the amount of label bound to the array. - View Dependent Claims (9, 10, 11, 12)
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13. A kit for detecting and/or quantifying a target polynucleotide sequence in a sample comprising:
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(α
) a probe comprising;
(i) a sequence segment (β
) having a 3′
end and a 5′
end, which sequence segment is complementary to at least a portion of the target polynucleotide,(ii) a first sequence segment (α
) covalently attached to one of the two ends of the sequence segment (β
),(iii) a second sequence segment (α
′
), which is at least partially complementary to the first sequence segment (α
), and is covalently attached to the other end of the sequence segment (β
),(iv) a means for attaching the probe to an array, the means being covalently linked to a free end of one of the first or second sequence segments (α
) or (α
′
),(v) a label attached to the end of the first or second sequence segments (α
) or (α
′
), which is not linked to the means for attaching the probe,the first and second sequence segments (α
) and (α
′
), respectively forming a duplex cleavage module containing a recognition site for a restriction endonuclease when the sequence segment (β
) does not hybridize with the target polynucleotide; and
(b) reagents necessary for implementing the detection/quantification of the target polynucleotide.
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14. An array comprising:
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(a) a solid phase; and
(b) a probe covalently attached to a surface of the solid phase comprising;
(i) a sequence segment (β
) having a 3′
end and a 5′
end, which sequence segment is complementary to at least a portion of the target polynucleotide,(ii) a first sequence segment (α
) covalently attached to one of the two ends of the sequence segment (β
),(iii) a second sequence segment (α
′
), which is at least partially complementary to the first sequence segment (α
), and is covalently attached to the other end of the sequence segment (β
),(iv) a means for covalently attaching the probe to a surface of the solid phase, the means being covalently linked to a free end of one of the first or second sequence segments (α
) or (α
′
),(v) a label attached to the end of the first or second sequence segments (α
) or (α
′
), which is not linked to the means for attaching the probe,the first and second sequence segments (α
) and (α
′
), respectively forming a duplex cleavage module containing a recognition site for a restriction endonuclease when the sequence segment (β
) does not hybridize with the target polynucleotide.
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Specification
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- Resources
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Current AssigneeFocusgenomics GmbH
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Original AssigneeFocusgenomics GmbH
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InventorsCharle, Christoph
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Application NumberUS10/497,130Publication NumberTime in Patent OfficeDaysField of SearchUS Class Current435/6CPC Class CodesC12Q 1/6837 using probe arrays or probe...C12Q 2525/131 incorporating a restriction...C12Q 2525/301 Hairpin oligonucleotides
