Method and system for sequencing polynucleotides

• US 20100130368A1
• Filed: 08/24/2009
• Published: 05/27/2010
• Est. Priority Date: 07/30/1998
• Status: Abandoned Application

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Osborne, Mark Allen, Balasubramanian, Shankar, Klenerman, David, Barnes, Colin
• Application Number:
  US12/583,658
• Publication Number:
  US 20100130368A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/0054 Means for coding or tagging the apparatus or the reagents
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/204 : specific length of the oligonucleotides
• CPC Class Codes: C12Q 2525/301 : Hairpin oligonucleotides
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/601 : being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

Provided herein is a method of determining a sequence of a target polynucleotide. The method can include the steps of a) providing a device including an array of relatively short polynucleotides and relatively long polynucleotides immobilised on a surface of a solid support, wherein the relatively long polynucleotides are fragments of the target polynucleotide and wherein the relatively long polynucleotides are separated by a distance of at least 10 nm, whereby parts of the relatively long polynucleotides that extend beyond the relatively short polynucleotides can be individually optically resolved; and b) determining the sequence of the target polynucleotide by detecting incorporation of nucleotides into strands complementary to the relatively long polynucleotide fragments using fluorescent labels associated with the incorporated nucleotides. Also provided is system for determining a sequence of a target polynucleotide. The system can include means for carrying out steps a) and b) of the above method.

Claims

1. A method of determining a sequence of a target polynucleotide comprising

a) providing a device comprising an array of relatively short polynucleotides and relatively long polynucleotides immobilised on a surface of a solid support, wherein the relatively long polynucleotides are fragments of the target polynucleotide and wherein the relatively long polynucleotides are separated by a distance of at least 10 nm, whereby parts of the relatively long polynucleotides that extend beyond the relatively short polynucleotides can be individually optically resolved;

and

b) determining the sequence of the target polynucleotide by detecting incorporation of nucleotides into strands complementary to the relatively long polynucleotide fragments using fluorescent labels associated with the incorporated nucleotides.

2. The method of claim 1 wherein density of the relatively short polynucleotides exceeds density of the relatively long polynucleotides by at least 100 fold.

3. The method according to claim 1 wherein the relatively long polynucleotides are linear polynucleotides and have both single stranded and double stranded portions.

4. The method according to claim 1 wherein each of the relatively long polynucleotides and each of the relatively short polynucleotides is immobilised by covalent bonding to the surface.

5. The method of claim 1 wherein the relatively long polynucleotides are separated by a distance of at least 100 nm.

6. The method of claim 1 wherein the relatively long polynucleotides are separated by a distance of at least 250 nm.

7. The method of claim 1 wherein the relatively short polynucleotides are in excess of the relatively long polynucleotides.

8. The method of claim 7 wherein providing the device comprises immobilizing the relatively short polynucleotides and the relatively long polynucleotides separately on the solid support, the relatively short polynucleotides being brought into contact with the solid support first.

9. The method of claim 7 wherein providing the device comprises bringing the relatively long polynucleotides and the relatively short polynucleotides into contact with the solid support in a single composition.

10. The method of claim 1 wherein the relatively long polynucleotides on the array comprise different sequences such that less than 50% of the relatively long polynucleotides are the same.

11. The method of claim 1 wherein the relatively long polynucleotides on the array comprise different sequences such that less than 30% of the relatively long polynucleotides are the same.

12. The method of claim 1 wherein the relatively long polynucleotides all comprise different sequences.

13. The method of claim 1 wherein the relatively long polynucleotides are 100 to 1000 nucleotides in length.

14. The method of claim 1 wherein density of the relatively long polynucleotides is 10⁶-10⁹ relatively long polynucleotides per cm².

15. The method of claim 14 wherein density of the relatively long polynucleotides is 10⁷-10⁸ relatively long polynucleotides per cm².

16. The method of claim 1 wherein the fluorescent labels are detected using total internal reflection fluorescence microscopy.

17. The method of claim 1 wherein the nucleotides carry a blocking group that prevents extension.

18. A system for determining a sequence of a target polynucleotide comprising

a) means for providing a device comprising an array of relatively short polynucleotides and relatively long polynucleotides immobilised on a surface of a solid support, wherein the relatively long polynucleotides are fragments of the target polynucleotide, and wherein the relatively long polynucleotides are separated by a distance of at least 10 nm, whereby parts of the relatively long polynucleotides that extend beyond the relatively short polynucleotides can be individually optically resolved;

and

b) means for determining the sequence of the target polynucleotide by detecting incorporation of nucleotides into strands complementary to the relatively long polynucleotides using fluorescent labels associated with the incorporated nucleotides.