Two-step hybridization and capture of a polynucleotide
First Claim
1. A method of capturing a target polynucleotide present in a sample, consisting essentially of the steps of:
- a) incubating a mixture in solution consisting essentially of a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe;
target hybridization complex formed by hybridization of the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe;
capture probe hybridization complex formed by hybridization of the immobilized probe and the capture probe; and
b) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe;
capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe;
capture probe;
target hybridization complex wherein the immobilized probe is hybridized to the capture probe which is hybridized to the target polynucleotide.
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Abstract
A method for capturing a target polynucleotide in a sample onto a solid support with an attached immobilized probe by using a capture probe and two different hybridization conditions, which preferably differ in temperature only, is disclosed. The two hybridization conditions control the order of hybridization, where the first hybridization conditions allow hybridization of the capture probe to the target polynucleotide, and the second hybridization conditions allow hybridization of the capture probe to the immobilized probe. The method may be used to detect the presence of a target polynucleotide in a sample by detecting the captured target polynucleotide or amplified target polynucleotide.
314 Citations
13 Claims
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1. A method of capturing a target polynucleotide present in a sample, consisting essentially of the steps of:
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a) incubating a mixture in solution consisting essentially of a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe;
target hybridization complex formed by hybridization of the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe;
capture probe hybridization complex formed by hybridization of the immobilized probe and the capture probe; andb) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe;
capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe;
capture probe;
target hybridization complex wherein the immobilized probe is hybridized to the capture probe which is hybridized to the target polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 11, 12, 13)
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7. A method of purifying a target polynucleotide present in a sample, consisting essentially of the steps of:
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a) incubating a mixture in solution consisting essentially of a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe;
target hybridization complex formed by hybridization of the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe;
capture probe hybridization complex formed by hybridization of the immobilized probe and the capture probe;b) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe;
capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe;
capture probe;
target hybridization complex wherein the immobilized probe is hybridized to the capture probe which is hybridized to the target polynucleotide; andc) purifying the immobilized probe;
capture probe;
target hybridization complex.
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8. A method of detecting a target polynucleolide present in a sample, consisting essentially of the steps of:
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a) incubating a mixture in solution consisting essentially of a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition that favors formation of a capture probe;
target hybridization complex formed by hybridization of the capture probe and the target polynucleotide, wherein the first hybridization condition disfavors formation of an immobilized probe;
capture probe hybridization complex formed by hybridization of the immobilized probe and the capture probe;b) then incubating the mixture in a second hybridization condition that favors formation of the immobilized probe;
capture probe hybridization complex, thereby capturing the target polynucleotide in an immobilized probe;
capture probe;
target hybridization complex wherein the immobilized probe is hybridized to the capture probe which is hybridized to the target polynucleotide;c) purifying the immobilized probe;
capture probe;
target hybridization complex; andd) detecting the target polynucleotide in the immobilized probe;
capture probe;
target hybridization complex. - View Dependent Claims (9, 10)
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Specification