Combinatorial decoding of random nucleic acid arrays

• US 9,163,283 B2
• Filed: 09/24/2013
• Issued: 10/20/2015
• Est. Priority Date: 05/20/1999
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Chee, Mark S., Walt, David R.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US14/035,611
• Publication Number:
  US 20140038832A1
• Time in Patent Office: 756 Days
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• 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 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• 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 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/161 Vesicles, e.g. liposome
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Abstract

Methods disclosed herein relate to identification of nucleotides in a nucleotide sequence.

Claims

1. A method of determining a nucleotide sequence of a captured nucleic acid, said method comprising the steps of:

a) providing a substrate comprising a first subpopulation and a second subpopulation of captured nucleic acids distributed thereon, wherein captured nucleic acids of the first subpopulation and captured nucleic acids of the second subpopulation comprise an anchor sequence adjacent to a target sequence, the anchor sequence of the first subpopulation of captured nucleic acids having the same sequence as the anchor sequence of the second subpopulation of captured nucleic acids and the target sequence of the first subpopulation of captured nucleic acids being different from the target sequence of the second subpopulation of captured nucleic acids,

b) hybridizing a first set of target probes to captured nucleic acids of the first subpopulation and captured nucleic acids of the second subpopulation, said first set of target probes comprising a first variable region, said first variable region comprising at least one detection nucleotide at a detection position, said detection nucleotide being able to basepair with a nucleotide of at least one of said different target sequences, wherein said first set of target probes comprises different groups, and wherein target probes from different groups comprise a different detection nucleotide at the same detection position and a label that distinguishes the detection nucleotide;

c) detecting the presence of said label;

d) associating the label with the identity of one or more nucleotides of at least one of said different target sequences;

e) dehybridizing said target probes from said captured nucleic acids;

f) hybridizing a second set of target probes to said first and second subpopulations of captured nucleic acids, wherein said second set of probes comprises a second variable region, said second variable region comprising a second detection nucleotide at a detection position, said second detection nucleotide being able to basepair with a nucleotide of at least one of said different target sequences, wherein said plurality of target probes from said second set comprises different groups, and wherein target probes from different groups of said second set comprise a different detection nucleotide at the same detection position and a label that distinguishes the second detection nucleotide, and

g) detecting the presence of a label in said second set of target probes.

2. The method of claim 1, wherein said first and second subpopulations of captured nucleic acids are attached to beads that are distributed within a channel.

3. The method of claim 1, wherein said target sequence of the first subpopulation of captured nucleic acids comprises a nucleic acid selected from the group consisting of:

a fragment of a gene, a regulatory sequence, genomic DNA, cDNA, RNA, a fragment of genomic DNA, a fragment of mRNA, and a copy of a genomic DNA fragment.

4. The method of claim 1, wherein said first set of target probes comprises at least four groups.

5. The method of claim 1, wherein said first variable region comprises two detection nucleotides.

6. The method of claim 5, wherein the two detection nucleotides are internal nucleotides of the variable region.

7. The method of claim 5, wherein the first of the two detection nucleotides is at an end of the variable region.

8. The method of claim 7, wherein the second of the two detection nucleotides is immediately adjacent to the first detection nucleotide.

9. The method of claim 8, wherein said variable region further comprises a plurality of random nucleotides immediately adjacent to the second detection nucleotide.

10. The method of claim 1, wherein said first variable region comprises a single detection nucleotide.

11. The method of claim 10, wherein the single detection nucleotide is an internal nucleotide of the variable region.

12. The method of claim 10, wherein the single detection nucleotide is at an end of the variable region.

13. The method of claim 12, wherein said variable region further comprises a plurality of random nucleotides immediately adjacent to the single detection nucleotide.

14. The method of claim 13, wherein said variable region further comprises a plurality of universal bases immediately adjacent to the plurality of random nucleotides.

15. The method of claim 14, wherein said plurality of universal bases consists of at least two but not more than five universal bases.

16. The method of claim 1, wherein said label in said first set of target probes is located at one end of said variable region.

17. The method of claim 1, wherein said label in said first set of target probes is located at the detection position.

18. The method of claim 1, wherein the target sequence comprises no more than 1000 nucleotides.

19. The method of claim 1, further comprising associating the label of the second set of target probes with the identity of one or more nucleotides of at least one of said different target sequences.

20. A method of determining a nucleotide sequence of a captured nucleic acid, said method comprising the steps of:

a) providing a substrate comprising a first subpopulation and a second subpopulation of captured nucleic acids distributed thereon, wherein captured nucleic acids of the first subpopulation and captured nucleic acids of the second subpopulation comprise an anchor sequence adjacent to a target sequence, the anchor sequence of the first subpopulation of captured nucleic acids having the same sequence as the anchor sequence of the second subpopulation of captured nucleic acids and the target sequence of the first subpopulation of captured nucleic acids being different from the target sequence of the second subpopulation of captured nucleic acids,

b) hybridizing a first set of target polynucleotide probes to captured nucleic acids of the first subpopulation and captured nucleic acids of the second subpopulation, said first set of target polynucleotide probes comprising a variable region, said variable region comprising at least one detection nucleotide at a detection position, said detection nucleotide being able to basepair with a nucleotide of at least one of said different target sequences, wherein said first set of target polynucleotide probes comprises different groups, and wherein target polynucleotide probes from different groups comprise a different detection nucleotide at the same detection position and a label that distinguishes the detection nucleotide;

c) detecting the presence of said label; and

d) associating the label with the identity of one or more nucleotides of at least one of said different target sequences.

21. The method of claim 20, further comprising:

e) dehybridizing said target polynucleotide probes from said captured nucleic acids;

f) hybridizing a second set of target polynucleotide probes to said first and second subpopulations of captured nucleic acids, wherein said second set of probes comprises a second variable region, said second variable region comprising a second detection nucleotide at a detection position, said second detection nucleotide being able to basepair with a nucleotide of at least one of said different target sequences, wherein said plurality of target polynucleotide probes from said second set comprises different groups, and wherein target polynucleotide probes from different groups of said second set comprise a different detection nucleotide at the same detection position and a label that distinguishes the second detection nucleotide, and

g) detecting the presence of a label in said second set of target polynucleotide probes.

22. The method of claim 21, further comprising associating the label of the second set of target polynucleotide probes with the identity of one or more nucleotides of at least one of said different target sequences.