Rolling circle replication of padlock probes
First Claim
Patent Images
1. A method comprising:
- i) providing a padlock probe for the target sequence, ii) forming a hybrid of the padlock probe with the target nucleic acid, and circularizing the padlock probe, iii) cutting the target nucleic acid at or near the target sequence, this step iii) being performed before, during or after step ii), and iv) effecting rolling circle replication of the padlock probe.
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Abstract
Rolling circle replication of a padlock primer is inhibited when it is hybridized 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 hybridizes 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 of assaying for a polyepitopic target by the use of two affinity probes each carrying an oligonucleotide tag and of a padlock probe for rolling circle replication in association with the two affinity probes
213 Citations
20 Claims
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1. A method comprising:
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i) providing a padlock probe for the target sequence, ii) forming a hybrid of the padlock probe with the target nucleic acid, and circularizing the padlock probe, iii) cutting the target nucleic acid at or near the target sequence, this step iii) being performed before, during or after step ii), and iv) effecting rolling circle replication of the padlock probe. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. An oligonucleotide suitable for use as a padlock probe for a target nucleic acid sequence, which oligonucleotide has 5′
- -end and 3′
-end sequences complementary to the target sequence;
a first site for recognition by a type IIS enzyme; and
a second site where at least one nucleotide residue and/or interucleotide bond is modified to protect the oligonucleotide from restriction by the type IIS enzyme. - View Dependent Claims (9, 10)
- -end and 3′
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11. A method comprising:
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i) cutting the target nucleic acid so as to create a target nucleic acid fragment having a 5′
-end target sequence and a 8′
-end target sequence,ii) providing a probe having two adjacent sequences complementary to the two target sequences, iii) forming a hybrid of the target nucleic acid fragment with the probe, and circularising the target nucleic acid fragment, and iv) effecting rolling circle replication of the circularised target nucleic acid fragment.
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12. A method comprising:
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i) providing a first padlock probe having 5′
-end and 8′
-end sequences which are complementary to the target sequence, and an intermediate sequence,ii) providing a second padlock probe having 5′
-end and 8′
-end sequences which are complementary to the intermediate sequence of the first padlock probe,iii) forming a hybrid by hybridising the first padlock probe to the target nucleic acid and hybridising the second padlock probe to the first padlock probe, and circularising both padlock probes, iv) purifying the hybrid, v) subjecting the hybrid to restriction thereby cutting the first padlock probe, and vi) effecting rolling circle replication of the second padlock. - View Dependent Claims (13, 14, 15)
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16. The method of 13, wherein in step v) restriction is effected by means of a type IIS enzyme.
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17. A kit for detecting a target sequence of a target nucleic acid, which kit comprises:
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a) a first padlock probe having 5′
-end and 3′
-end sequences which are complementary to the target sequence, and an intermediate sequence, andb) a second padlock probe having 5′
-end and 3′
-end sequences which are complementary to the intermediate sequence of the first probe.
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18. A kit for assaying for a polyepitopic target which kit 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 terminal second polynucleotide sequence, and c) a padlock probe having 5′
-end and 3′
-end sequences which are complementary to the first polynucleotide sequence, and an intermediate sequence which is complementary to the second polynucleotide sequence.- View Dependent Claims (19)
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20. A method comprising:
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providing a first affinity probe for the 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 terminal second polynucleotide sequence, a padlock probe having 5′
-end and 3′
-end sequences which are complementary to the first polynucleotide sequence, and an intermediate sequence which is complementary to the second polynucleotide sequence;
binding the first affinity probe to the target;
binding the second affinity probe to the target;
hybridising the padlock probe to the first polynucleotide sequence and to the second polynucleotide sequence;
circularising the padlock probe; and
using the second polynucleotide sequence as a primer to effect rolling circle amplification of the padlock probe.
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Specification