Methods and compositions for tagging and identifying polynucleotides

• US 8,476,018 B2
• Filed: 05/08/2012
• Issued: 07/02/2013
• 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:
  US13/466,883
• Publication Number:
  US 20130065770A1
• Time in Patent Office: 420 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 method of estimating the number of target polynucleotides in a sample, comprising:

ligating a discriminatory oligonucleotide tag to a complex nucleic acid sample comprising multiple molecular species so that substantially every target polynucleotide receives a tag that comprises a unique nucleotide sequence, to produce a tagged sample;

amplifying the target polynucleotides in said tagged sample; and

determining the number of different tags that are present in the amplified target polynucleotides, thereby providing an estimate of the number of target polynucleotides that are amplified.

2. The method of claim 1, wherein said amplifying uses a primer that hybridizes to said oligonucleotide tag.

3. The method of claim 1, wherein said target polynucleotide is a fragment of genomic DNA.

4. The method of claim 1, wherein said determining is done by hybridizing the amplified target polynucleotides to an array.

5. The method of claim 1, wherein said determining is done by DNA sequencing.

6. The method of claim 1, further comprising sequencing the amplified target polynucleotides.

7. The method of claim 1, wherein said oligonucleotide tag comprises a degenerate discriminatory sequence of 2 to 18 nucleotides.

8. The method of claim 1, wherein said target polynucleotides are isolated prior to amplification.

9. The method of claim 8, wherein said target polynucleotides are isolated by hybridizing said pooled sample to a probe that is immobilized to a solid support.

10. The method of claim 8, wherein said target polynucleotides are isolated by hybridizing said pooled sample to a biotinylated probe that is captured by a solid support.

11. The method of claim 8, wherein said isolating is done by exonuclease digestion of non-circularized probes.

12. The method of claim 8, wherein said isolating is done by template-driven ligation.

13. The method of claim 8, wherein said isolating is done by template-driven strand extension.