Nanogrid rolling circle DNA sequencing

Patent metadata

Field	Value
Publication number	US-9624538-B2
Application number	US-12054108-A
Country	US
Kind code	B2
Filing date	May 14, 2008
Priority date	Nov 14, 2005
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

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.

First claim

What is claimed:

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.

2. The method of claim 1 , wherein the concatemer has a cross-sectional diameter of at least 200 nanometers.

3. The method of claim 1 , wherein the concatemer has a cross-sectional diameter of at least 300 nanometers.

4. The method of claim 1 , wherein the polynucleotide is DNA.

5. The method of claim 1 , wherein one concatemer is immobilized at the plurality of specific functionalized regions.

6. The method of claim 1 , wherein the array has at least 100 specific functionalized regions.

7. The method of claim 6 , wherein one concatemer is immobilized at each of the plurality of specific functionalized regions.

8. The method of claim 6 , wherein at least 90 of the specific functionalized regions each contains one immobilized concatemer.

9. The method of claim 1 , wherein the array has at least 1000 specific functionalized regions.

10. The method of claim 1 , wherein the array has at least 10,000 specific functionalized regions.

11. The method of claim 1 , wherein sequencing the immobilized concatemer is performed by fluorescent in situ sequencing.

12. The method of claim 1 , wherein the array is a nanoarray.

13. The method of claim 1 , wherein optical magnification is used to sequence the immobilized concatemer.

14. The method of claim 1 , wherein immobilization is performed by hybridization.

15. The method of claim 1 , wherein immobilization is performed by an interaction selected from the group consisting of biotin-avidin capture, biotin-streptavidin capture, NHS-ester capture, thioether linkage, static charge interactions and van der Waals forces.

16. 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.

17. The method of claim 16 , wherein clonally amplifying is performed by a method selected from the group consisting of rolling circle amplification, multiple displacement amplification, thermophilic helicase-dependent amplification and bridge PCR.

18. The method of claim 17 , wherein rolling circle amplification is selected from the group consisting of hyperbranched rolling circle amplification, padlock probe rolling circle amplification and linear rolling circle amplification.

19. The method of claim 1 , wherein immobilization is performed by biotin-avidin capture or NHS-ester capture.

Assignees

• Church George M
• Porreca Gregory J
• Shendure Jay
• Rosenbaum Abraham Meir
• Harvard College

Inventors

• Church George M
• Porreca Gregory J
• Shendure Jay
• Rosenbaum Abraham Meir

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2531/125

  Rolling circle · CPC title

• C12Q2565/513

  characterised by the pattern of the arrayed oligonucleotides · CPC title

• C12Q2563/155

  Particles of a defined size, e.g. nanoparticles · CPC title