Methods and compositions for tagging and identifying polynucleotides

• US 8,470,996 B2
• Filed: 05/08/2012
• Issued: 06/25/2013
• Est. Priority Date: 02/10/2005
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/466,894
• Publication Number:
  US 20130065793A1
• Time in Patent Office: 413 Days
• Field of Search: None
• US Class Current: 536/24.3
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• 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 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• 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: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention provides methods and compositions for attaching oligonucleotide tags to polynucleotides for the purpose of carrying out analytical assays in parallel and for decoding the oligonucleotide tags of polynucleotides selected in such assays. Words, or subunits, of oligonucleotide tags index submixtures in successively more complex sets of submixtures (referred to herein as “tiers” of submixtures) that a polynucleotide goes through while successive words are added to a growing tag. By identifying each word of an oligonucleotide tag, a series of submixtures is identified including the first submixture that contains only a single polynucleotide, thereby providing the identity of the selected polynucleotide. The analysis of the words of an oligonucleotide tag can be carried out in parallel, e.g. by specific hybridization of the oligonucleotide tag to its tag complement on an addressable array; or such analysis can be carried out serially by successive specific hybridizations of labeled word complements, or the like.

Claims

1. A composition of matter comprising:

a) a solid-support; and

b) a plurality of genomic fragments attached to the solid support in a way that is not spatially addressable, wherein;

i. said genomic fragments are derived from a plurality of different sources;

ii each of said genomic fragments comprises an oligonucleotide tag that comprises a unique nucleotide sequence that indicates from which source a genomic fragment is derived and, separate from said unique nucleotide sequence, a binding site for a sequencing primer; and

iii. said genomic fragments correspond to one or more selected portions of a genome that have been isolated from a pooled genomic sample by specific hybridization.

2. The composition of matter of claim 1, wherein said genomic fragments are covalently attached to said solid support by their 5′

end.

3. The composition of matter of claim 1, wherein said genomic fragments are covalently attached to said solid support by their 3′

end.

4. The composition of matter of claim 1, wherein said genomic fragments are non-covalently attached to said solid support.

5. The composition of matter of claim 1, wherein said genomic fragments are attached to a planar surface of said solid support.

6. The composition of matter of claim 1, wherein the identities of said genomic fragments are discernible by sequence analysis.

7. The composition of matter of claim 1, wherein said solid support comprises raised regions or etched trenches.

8. The composition of matter of claim 1, wherein said solid support is in the form of one or more beads.

9. The composition of matter of claim 1, wherein said one or more selected portions of a genome comprise a portion of a gene.

10. The composition of matter of claim 1, wherein said selected portions of said genome contains a nucleotide sequence that varies in said different sources.

11. The composition of matter of claim 1, wherein said genomic fragments comprise fragments in the range of 100 to 300 nucleotides in length.

12. The composition of matter of claim 1, wherein said plurality of different sources are different human individuals.

13. The composition of matter of claim 1, wherein said plurality of different sources comprise 50 to 10,000 different sources.

14. The composition of matter of claim 1, wherein said unique nucleotide sequence is from 2 to 18 nucleotides in length.