Rolling circle replication of padlock probes
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Abstract
Rolling circle replication of a padlock primer is inhibited when it is hybridised to a target nucleic acid that is long or circular. The invention provides methods of addressing this problem including cutting the target nucleic acid near or preferably at the site which hybridises with the padlock probe, whereby a 3′-end of the cut target nucleic acid acts as a primer for rolling circle replication of the padlock probe. Also included is a method for assaying for a polyepitopic target by the use of two affinity probes each carrying an oligonucleotide tag and of padlock probe for rolling circle replication in association with the two affinity probes.
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Citations
12 Claims
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1. (canceled)
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2. A method comprising:
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providing a first affinity probe for a target which first affinity probe carries a polynucleotide chain including a first polynucleotide sequence, a second affinity probe for the target which second affinity probe carries a polynucleotide chain including a second polynucleotide sequence, a padlock probe provided in two parts wherein the first part of the padlock probe has a 5′
end which is complimentary to the first polyoligonucleotide and a 3′
end complementary to the second polyoligonucleotide, and the second part of the padlock probe has a 3′
end which is complementary to the first polyoligonucleotide and a 5′
end complementary to the second polyoligonucleotide;
binding the first affinity probe to the target;
binding the second affinity probe to the target;
hybridizing the padlock probe provided in two parts;
circularizing the padlock probe; and
effecting amplification of the padlock probe. - View Dependent Claims (3, 4, 5)
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6. A kit for assaying for a polyepitopic target which comprises:
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a) a first affinity probe for the target which first affinity probe carries a polynucleotide chain including a first polynucleotide sequence, b) a second affinity probe for the target which second affinity probe carries a polynucleotide chain including a second polynucleotide sequence, and c) a padlock probe provided in two parts wherein the first part of the padlock probe has a 5′
end which is complementary to the first polyoligonucleotide and a 3′
end complementary to the second polyoligonucleotide, and the second part of the padlock probe has a 3′
end which is complementary to the first polyoligonucleotide and a 5′
end complementary to the second polyoligonucleotide.
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7. (canceled)
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8. A method comprising:
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providing a first affinity probe for a target which first affinity probe carries a polynucleotide chain including a first polynucleotide sequence, a second affinity probe for the target which second affinity probe carries a polynucleotide chain including a second polynucleotide sequence, two or more padlock probes, each padlock probe being provided in two parts, wherein a first padlock probe has 5′
end and 3′
end sequences that are complementary to the target sequence, and an intermediate sequence, and a second padlock probe has 5′
end and 3′
end sequences which are complementary to the intermediate sequence of the first padlock probe,binding the first affinity probe to the target;
binding the second affinity probe to the target;
hybridizing the first padlock probe to the target sequence and hybridizing the second padlock probe to the first padlock probe;
circularizing the padlock probes; and
effecting amplification of the padlock probes. - View Dependent Claims (9, 10, 11)
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12. A kit for assaying for a polyepitopic target which comprises:
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a) a first affinity probe for the target which first affinity probe carries a polynucleotide chain including a first polynucleotide sequence, b) a second affinity probe for the target which second affinity probe carries a polynucleotide chain including a second polynucleotide sequence, and c) two or more padlock probes, each padlock probe being provided in two parts, wherein a first padlock probe has 5′
end and 3′
end sequences which are complementary to the first polynucleotide sequence, and an intermediate sequence, and a second padlock probe has 5′
end and 3′
end sequences which are complementary to the intermediate sequence of the first padlock probe and the second polynucleotide sequence.
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Specification