Nanogrid rolling circle DNA sequencing
First Claim
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1. A method of sequencing a polynucleotide immobilized on an array comprising:
- synthesizing a concatemer of a polynucleotide by rolling circle amplification;
immobilizing the concatemer to the array, wherein the array comprises a substrate having a plurality of specific functionalized regions attached to the substrate in a pattern forming an ordered array, wherein each specific functionalized region comprises an inner spot functionalized with a first group which serves to capture the concatemer and an outer spot functionalized with a second group which serves as an attachment point for an amplification primer, wherein the concatermer is immobilized to the inner spot, and wherein the concatemer has a cross-sectional diameter greater than the diameter of the inner spot; and
sequencing the immobilized concatemer.
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Abstract
The present invention relates to methods for sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size, including synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter greater than the diameter of a specific region, immobilizing the concatemer to the specific region to make an immobilized concatemer, and sequencing the immobilized concatemer.
57 Citations
19 Claims
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1. A method of sequencing a polynucleotide immobilized on an array comprising:
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synthesizing a concatemer of a polynucleotide by rolling circle amplification; immobilizing the concatemer to the array, wherein the array comprises a substrate having a plurality of specific functionalized regions attached to the substrate in a pattern forming an ordered array, wherein each specific functionalized region comprises an inner spot functionalized with a first group which serves to capture the concatemer and an outer spot functionalized with a second group which serves as an attachment point for an amplification primer, wherein the concatermer is immobilized to the inner spot, and wherein the concatemer has a cross-sectional diameter greater than the diameter of the inner spot; and sequencing the immobilized concatemer. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 19)
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16. A method of sequencing a polynucleotide immobilized on an comprising:
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synthesizing a concatemer of a polynucleotide by rolling circle amplification immobilizing the concatemer to the array, wherein the array comprises a substrate having a plurality of specific functionalized regions attached to the substrate in a pattern forming an ordered array, wherein each specific functionalized region comprises an inner spot functionalized with a first group which serves to capture the concatemer and an outer spot functionalized with a second group which serves as an attachment point for an amplification primer, wherein the concatermer is immobilized to the inner spot, and wherein the concatemer has a cross-sectional diameter greater than the diameter of the inner spot; clonally amplifying the immobilized concatemer to make a clonally amplified concatemer; and sequencing the clonally amplified concatemer. - View Dependent Claims (17, 18)
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Specification