NANOGRID ROLLING CIRCLE DNA SEQUENCING

• US 20090018024A1
• Filed: 05/14/2008
• Published: 01/15/2009
• Est. Priority Date: 11/14/2005
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Rosenbaum, Abraham Meir, CHURCH, George M., Porreca, Gregory J., Shendure, Jay
• Granted Patent: US 9,624,538 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides

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.

Claims

1. A method of sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size comprising:

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.

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 specific region.

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

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

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

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

10. The method of claim 1, wherein the array has at least 10,000 specific 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 array having a plurality of specific regions each having a defined diameter size comprising:

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;

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. A method of sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size of less than 300 nanometers comprising:

synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter of greater than 300 nanometers;

immobilizing the concatemer to the specific region to make an immobilized concatemer; and

sequencing the immobilized concatemer.