Methods, kits and compositions for detecting and quantitating target sequences
First Claim
1. A method of forming a probe triplex, comprising contacting a sample containing target nucleic acid, to at least one probe set comprising three probes, wherein the first and second probes comprise a probing segment and a PNA arm segment, and wherein the third probe comprises a PNA arm segment and wherein the probing segment of the first probe hybridizes to a first site of hybridization on the target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe, under suitable hybridizing conditions, such that the arm segments of the first, second and third probes form the PNA probe triplex only in the presence of the target nucleic acid.
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Abstract
The present invention relates to methods, kits and compositions suitable for the improved detection, quantitation and analysis of nucleic acid target sequences using probe-based hybridization assays.
162 Citations
61 Claims
- 1. A method of forming a probe triplex, comprising contacting a sample containing target nucleic acid, to at least one probe set comprising three probes, wherein the first and second probes comprise a probing segment and a PNA arm segment, and wherein the third probe comprises a PNA arm segment and wherein the probing segment of the first probe hybridizes to a first site of hybridization on the target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe, under suitable hybridizing conditions, such that the arm segments of the first, second and third probes form the PNA probe triplex only in the presence of the target nucleic acid.
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31. A method for detecting target nucleic acid present in a sample, comprising forming a PNA probe triplex comprising contacting a sample containing target nucleic acid, to at least one probe set comprising three probes, wherein the first and second probes comprise a probing segment and a PNA arm segment, and wherein the third probe comprises a PNA arm segment and wherein the probing segment of the first probe hybridizes to a first site of hybridization on the target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe, under suitable hybridizing conditions, such that the arm segments of the first, second and third probes form the PNA probe triplex only in the presence of the target nucleic acid;
- and detecting the PNA probe triplex.
- View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A method for quantifying target nucleic acid present in a sample, comprising forming a PNA probe triplex comprising contacting a sample containing target nucleic acid, to at least one probe set comprising three probes, wherein the first and second probes comprise a probing segment and a PNA arm segment, and wherein the third probe comprises a PNA arm segment and wherein the probing segment of the first probe hybridizes to a first site of hybridization on the target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe, under suitable hybridizing conditions, such that the PNA arm segments of the first, second and third probes form the PNA probe triplex, wherein the quantity of target nucleic acid is determined by target-dependent signal.
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50. A probe set comprising:
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a) a first and second nucleobase probe wherein each probe has a probing segment and a PNA arm segment, such that the probing segment of the first probe hybridizes to a first site of hybridization on a target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe; and
b) a third nucleobase probe having a PNA arm segment such that the arm segments of the first, second and third probes form a PNA probe triplex under suitable hybridizing conditions when the first and second probes hybridize to the first and second sites of hybridization, respectively. - View Dependent Claims (51, 52, 53, 54, 55, 56)
a) at least one probe set of claim 50; and
b) reagents suitable for detecting the presence or amount of target nucleic acid in the sample.
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56. The kit of claim 55, wherein the reagent of b) includes an antibody specific for detecting the presence or amount of the PNA probe triplex.
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57. A probe triplex/target nucleic acid complex comprising:
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a) a target nucleic acid;
b) a first and second nucleobase probe wherein each probe has a probing segment and a PNA arm segment, such that the probing segment of the first probe hybridizes to the a first site of hybridization on a target nucleic acid which is in close proximity to a second site of hybridization of the probing segment of the second probe; and
c) a third nucleobase probe having a PNA arm segment such that the arm segments of the first, second and third probes form a PNA probe triplex under suitable hybridizing conditions when the first and second probes hybridize to the first and second sites of hybridization, respectively.
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58. A PNA polymer comprising;
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a) a first PNA segment having a first end;
b) a second PNA segment having a first end, wherein the first and second PNA segments participate in the formation of a PNA triplex structure upon interaction with a target sequence of a third PNA containing segment;
c) a flexible linkage that covalently links the first end of the first PNA segment to the first end of the second PNA segment;
d) at least one donor moiety linked to either of the first PNA segment or the second PNA segment; and
e) at least one acceptor moiety linked to the other of either of the first PNA segment or the second PNA segment. - View Dependent Claims (59, 60, 61)
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Specification