Padlock probe detection
First Claim
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1. A method for the detection of a second polynucleotide, comprising:
- (A) providing a first polynucleotide, wherein the first polynucleotide comprises;
a segment, the segment comprising the complement of at least the terminal four nucleotides of the 5'"'"' and 3'"'"' termini of the second polynucleotide such that the 3'"'"' and 5'"'"' termini of the second polynucleotide abut to each other upon hybridization of the first and second polynucleotides, wherein the first polynucleotide has a first ligand attached on one side of the segment and a first ligand attached on the other side of the segment;
(B) placing the first polynucleotide in contact with the second polynucleotide under hybridizing conditions;
(C) immobilizing the first polynucleotide by contacting with a solid surface comprising a second ligand that binds to the first ligands of the first polynucleotide; and
(D) detecting any second polynucleotide immobilized in a complex with the first polynucleotide.
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Abstract
The present invention relates to the detection of a polynucleotide using a padlock probe, whereby a hybridization process is combined with ligation to irreversibly capture polynucleotides of interest. The invention relates both to the padlock probe itself, as well as to methods of use, which allow for varying levels of sensitivity due to amplification of signal processes built into the system. Additionally, preferred formats are disclosed for use of the padlock probes, including the use of microfluidics.
175 Citations
20 Claims
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1. A method for the detection of a second polynucleotide, comprising:
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(A) providing a first polynucleotide, wherein the first polynucleotide comprises;
a segment, the segment comprising the complement of at least the terminal four nucleotides of the 5'"'"' and 3'"'"' termini of the second polynucleotide such that the 3'"'"' and 5'"'"' termini of the second polynucleotide abut to each other upon hybridization of the first and second polynucleotides, wherein the first polynucleotide has a first ligand attached on one side of the segment and a first ligand attached on the other side of the segment;(B) placing the first polynucleotide in contact with the second polynucleotide under hybridizing conditions; (C) immobilizing the first polynucleotide by contacting with a solid surface comprising a second ligand that binds to the first ligands of the first polynucleotide; and (D) detecting any second polynucleotide immobilized in a complex with the first polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 19)
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9. A method for the detection of a second polynucleotide, comprising:
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(A) providing a first polynucleotide, wherein the first polynucleotide comprises; (i) a first ligand attached to each of its termini; and (ii) at least one first segment comprising the complement of at least the terminal four nucleotides of each of the 5'"'"' and 3'"'"' or the 3'"'"' and 5'"'"' termini of a first probe and a second probe, respectively; (B) providing the first probe comprising a second segment located at one of its termini and having at least four nucleotides of the complement of the second polynucleotide; (C) providing the second probe comprising a third segment located at one of its termini and having at least four nucleotides of the complement of the second polynucleotide, with the proviso that the second and third segments are different and are the complements of contiguous segments of the second polynucleotide; (D) contacting the first polynucleotide with a solid surface or a solid surface comprising a second ligand that binds to the first ligands of the first polynucleotide, thereby immobilizing the first polynucleotide; (E) contacting the first and second probes with a second polynucleotide under conditions that promote hybridization between complementary nucleic acids, forming a probe-second polynucleotide complex; (F) joining the termini of the hybridized first and second probes of the probe-second polynucleotide complex with a joining agent; (G) contacting the joined first and second probes with the first polynucleotide under conditions that promote hybridization between complementary nucleic acids, forming a probe-first polynucleotide complex; (H) joining the termini of the hybridized first and second probes of the probe-first polynucleotide complex with a joining agent; (I) washing the probe-first polynucleotide complex with a wash fluid, thereby removing unhybridized joined first and second probe; and (J) detecting the second polynucleotide by detecting a label or third ligand attached to the first or second probe, wherein the third ligand is recognized by a fourth ligand that includes a label. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 20)
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10. The method of claim 10, wherein steps (E), (F), (G), and (H) are combined.
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