Nanogrid rolling circle DNA sequencing
AssigneePresident and Fellows of Harvard College
InventorsGeorge M. Church, Gregory J. Porreca, Jay Shendure, Abraham Meir Rosenbaum
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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.
Claims19
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.
A method of sequencing a polynucleotide immobilized on an 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;
- clonally amplifying the immobilized concatemer to make a clonally amplified concatemer;
- and
- sequencing the clonally amplified concatemer.
Dependencies are inferred from the claim text.