Methods and composition for detecting targets
First Claim
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1. A method for detecting the presence or absence of at least one target nucleic acid sequence in a sample comprising:
- forming a ligation reaction composition comprising the sample, and a ligation probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′
primer-specific portion, wherein the 5′
primer-specific portion comprises a sequence, and (b) at least one second probe, comprising a target-specific portion and a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;
forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′
primer-specific portion, the target-specific portions, and the 3′
primer-specific portion;
forming at least one amplification reaction composition comprising;
at least a portion of the test composition;
a polymerase;
a double-stranded-dependent specific label, wherein the double-stranded-dependent label has a first detectable signal value when the double-stranded-dependent label is not exposed to double-stranded nucleic acid; and
at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′
primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3′
primer-specific portion of the ligation product;
subjecting the at least one amplification reaction composition to at least one amplification reaction; and
detecting a second detectable signal value at least one of during and after the at least one amplification reaction, wherein a threshold difference between the first detectable signal value and the second detectable signal value indicates the presence of the target nucleic acid sequence, and wherein no threshold difference between the first detectable signal value and the second detectable signal value indicates the absence of the target nucleic acid sequence.
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Abstract
The present invention relates to methods and kits for detecting the presence or absence of (or quantitating) target nucleic acid sequences using ligation and amplification.
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Citations
55 Claims
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1. A method for detecting the presence or absence of at least one target nucleic acid sequence in a sample comprising:
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forming a ligation reaction composition comprising the sample, and a ligation probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′
primer-specific portion, wherein the 5′
primer-specific portion comprises a sequence, and (b) at least one second probe, comprising a target-specific portion and a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;
forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′
primer-specific portion, the target-specific portions, and the 3′
primer-specific portion;
forming at least one amplification reaction composition comprising;
at least a portion of the test composition;
a polymerase;
a double-stranded-dependent specific label, wherein the double-stranded-dependent label has a first detectable signal value when the double-stranded-dependent label is not exposed to double-stranded nucleic acid; and
at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′
primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3′
primer-specific portion of the ligation product;
subjecting the at least one amplification reaction composition to at least one amplification reaction; and
detecting a second detectable signal value at least one of during and after the at least one amplification reaction, wherein a threshold difference between the first detectable signal value and the second detectable signal value indicates the presence of the target nucleic acid sequence, and wherein no threshold difference between the first detectable signal value and the second detectable signal value indicates the absence of the target nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method for detecting the presence or absence of at least one target nucleic acid sequence in a sample comprising:
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forming a ligation reaction composition comprising the sample, and a ligation probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′
primer-specific portion, wherein the 5′
primer-specific portion comprises a sequence, and (b) at least one second probe, comprising a target-specific portion and a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;
forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′
primer-specific portion, the target-specific portions, and the 3′
primer-specific portion;
forming at least one amplification reaction composition comprising;
at least a portion of the test composition, a polymerase, a double-stranded-dependent label; and
at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′
primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3′
primer-specific portion of the ligation product;
subjecting the at least one amplification reaction composition to at least one amplification reaction; and
detecting the presence or absence of the target nucleic acid sequence by monitoring a signal at least one of during and after the at least one amplification reaction. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method for detecting the presence or absence of at least one target nucleic acid sequence in a sample comprising:
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(a) forming at least one reaction composition comprising;
the sample;
a ligation probe set for the target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′
primer-specific portion, wherein the 5′
primer-specific portion comprises a sequence and (b) at least one second probe, comprising a target-specific portion and a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;
a polymerase;
a double-stranded-dependent label, wherein the double-stranded-dependent label has a first detectable signal value when the double-stranded-dependent label is not exposed to double-stranded nucleic acid; and
at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′
primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3′
primer-specific portion of the ligation product;
(b) subjecting the reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′
primer-specific portion, the target-specific portions, and the 3′
primer-specific portion;
(c) after the at least one cycle of ligation, subjecting the reaction composition to at least one amplification reaction; and
(d) detecting a second detectable signal value at least one of during and after the at least one amplification reaction, wherein a threshold difference between the first detectable signal value and the second detectable signal value indicates the presence of the target nucleic acid sequence, and wherein no threshold difference between the first detectable signal value and the second detectable signal value indicates the absence of the target nucleic acid sequence. - View Dependent Claims (28, 29, 30, 31, 32)
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33. A method for detecting the presence or absence of at least one target nucleic acid sequence in a sample comprising:
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(a) forming at least one reaction composition comprising;
the sample;
a ligation probe set for the target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′
primer-specific portion, wherein the 5′
primer-specific portion comprises a sequence and (b) at least one second probe, comprising a target-specific portion and a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;
a polymerase;
a double-stranded-dependent label; and
at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′
primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3′
primer-specific portion of the ligation product;
(b) subjecting the reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′
primer-specific portion, the target-specific portions, and the 3′
primer-specific portion;
(c) after the at least one cycle of ligation, subjecting the reaction composition to at least one amplification reaction; and
(d) detecting the presence or absence of the target nucleic acid sequence by monitoring a signal at least one of during and after the at least one amplification reaction. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A kit for detecting at least one target nucleic acid sequence in a sample comprising:
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(a) a ligation probe set for each target nucleic acid sequence, the probe set comprising (i) at least one first probe, comprising a target-specific portion, a 5′
primer-specific portion, wherein the 5′
primer-specific portion comprises a sequence, and(ii) at least one second probe, comprising a target-specific portion, a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence,wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target nucleic acid sequence; and
(b) a double-stranded-dependent label. - View Dependent Claims (50)
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51. A method for detecting the presence or absence of at least one target nucleic acid sequence in a sample comprising:
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forming a ligation reaction composition comprising the sample;
a ligation probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion, and (b) at least one second probe, comprising a target-specific portion, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence; and
poly-deoxy-inosinic-deoxy-cytidylic acid;
forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′
primer-specific portion, the target-specific portions, and the 3′
primer-specific portion; and
detecting the presence or absence of the ligation product to detect the presence or absence of the at least one target nucleic acid sequence.
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52. A method for detecting the presence or absence of at least one target nucleic acid sequence in a sample comprising:
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forming a ligation reaction composition comprising the sample, poly-deoxy-inosinic-deoxy-cytidylic acid, and a ligation probe set for each target nucleic acid sequence, the probe set comprising (a) at least one first probe, comprising a target-specific portion and a 5′
primer-specific portion, wherein the 5′
primer-specific portion comprises a sequence, and (b) at least one second probe, comprising a target-specific portion and a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence, wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target sequence;
forming a test composition by subjecting the ligation reaction composition to at least one cycle of ligation, wherein adjacently hybridizing complementary probes are ligated to one another to form a ligation product comprising the 5′
primer-specific portion, the target-specific portions, and the 3′
primer-specific portion;
forming at least one amplification reaction composition comprising;
at least a portion of the test composition;
a polymerase; and
at least one primer set, the primer set comprising (i) at least one first primer comprising the sequence of the 5′
primer-specific portion of the ligation product, and (ii) at least one second primer comprising a sequence complementary to the sequence of the 3′
primer-specific portion of the ligation product;
subjecting the at least one amplification reaction composition to at least one amplification reaction; and
detecting the presence or absence of the target nucleic acid sequence by detecting whether the at least one amplification reaction results in amplification product from ligation product.
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53. A kit for detecting at least one target nucleic acid sequence in a sample comprising:
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(a) a ligation probe set for each target nucleic acid sequence, the probe set comprising (i) at least one first probe, comprising a target-specific portion, a 5′
primer-specific specific portion, wherein the 5′
primer-specific portion comprises a sequence, and(ii) at least one second probe, comprising a target-specific portion, a 3′
primer-specific portion, wherein the 3′
primer-specific portion comprises a sequence,wherein the probes in each set are suitable for ligation together when hybridized adjacent to one another on a complementary target nucleic acid sequence; and
(b) a buffer comprising poly-deoxy-inosinic-deoxy-cytidylic acid. - View Dependent Claims (54)
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55. A composition for a ligation reaction comprising a ligase and poly-deoxy-inosinic-deoxy-cytidylic acid.
Specification