Directed enrichment of genomic DNA for high-throughput sequencing
First Claim
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1. A method for amplifying a target polynucleotide, comprising:
- a) providing a plurality of oligonucleotide primer pairs, wherein each oligonucleotide primer pair is located at a discrete position on a microarray, and wherein each oligonucleotide primer in each oligonucleotide primer pair comprises at least one cleavable linkage;
b) hybridizing a target polynucleotide to at least one primer in at least one oligonucleotide primer pair on the microarray, thereby producing a microarray comprising at least one primer pair comprising at least one primer hybridized to a target polynucleotide;
c) embedding the microarray of step b) into a capturing support;
d) separating the at least one primer oligonucleotide pair comprising at least one primer hybridized to a target polynucleotide from the microarray by cleaving the at least one cleavable linkage in each primer, thereby releasing the at least one oligonucleotide primer pair comprising at least one primer hybridized to a target polynucleotide from the microarray, wherein the at least one oligonucleotide primer pair comprising at least one primer hybridized to a target polynucleotide is captured in the capturing support; and
e) amplifying the target polynucleotide under conditions in which the at least one oligonucleotide primer pair comprising at least one primer hybridized to the target polynucleotide amplifies the target polynucleotide by polymerase chain reaction, whereby an amplified target polynucleotide is produced;
thereby amplifying a target polynucleotide.
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Abstract
The present invention provides microarrays of oligonucleotide primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or more microarrays, and methods to use the captured oligonucleotide primer pairs, such as for amplification of a target polynucleotide sequence. In addition, methods of using a microarray to isolate, purify and/or amplify a target polynucleotide are provided.
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Citations
39 Claims
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1. A method for amplifying a target polynucleotide, comprising:
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a) providing a plurality of oligonucleotide primer pairs, wherein each oligonucleotide primer pair is located at a discrete position on a microarray, and wherein each oligonucleotide primer in each oligonucleotide primer pair comprises at least one cleavable linkage; b) hybridizing a target polynucleotide to at least one primer in at least one oligonucleotide primer pair on the microarray, thereby producing a microarray comprising at least one primer pair comprising at least one primer hybridized to a target polynucleotide; c) embedding the microarray of step b) into a capturing support; d) separating the at least one primer oligonucleotide pair comprising at least one primer hybridized to a target polynucleotide from the microarray by cleaving the at least one cleavable linkage in each primer, thereby releasing the at least one oligonucleotide primer pair comprising at least one primer hybridized to a target polynucleotide from the microarray, wherein the at least one oligonucleotide primer pair comprising at least one primer hybridized to a target polynucleotide is captured in the capturing support; and e) amplifying the target polynucleotide under conditions in which the at least one oligonucleotide primer pair comprising at least one primer hybridized to the target polynucleotide amplifies the target polynucleotide by polymerase chain reaction, whereby an amplified target polynucleotide is produced; thereby amplifying a target polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 39)
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33. A method for amplifying a target polynucleotide, comprising:
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a) providing an oligonucleotide primer located at a discrete position on a microarray, wherein the oligonucleotide primer comprises at least one cleavable linkage; b) hybridizing a target polynucleotide to the oligonucleotide primer on the microarray, wherein the target polynucleotide comprises a universal adapter ligated to each end of the target polynucleotide, and wherein each universal adapter comprises a universal primer sequence, thereby producing a microarray comprising an oligonucleotide primer hybridized to a target polynucleotide; c) extending the oligonucleotide primer hybridized to the target polynucleotide, thereby producing a first strand primer extension product located at a discrete location on the microarray; d) removing the target polynucleotide from the first strand primer extension product located at a discrete location on the microarray; e) embedding the first strand primer extension product located at a discrete location on the microarray into a capturing support, wherein the capturing support comprises a universal primer; f) amplifying the target polynucleotide in the capturing support under conditions in which the oligonucleotide primer hybridized to the target polynucleotide and the universal primer amplifies the target polynucleotide by polymerase chain reaction, whereby an amplified target polynucleotide is produced; thereby amplifying a target polynucleotide. - View Dependent Claims (34, 35, 36, 37, 38)
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Specification