METHODS AND COMPOSITIONS FOR TAGGING AND IDENTIFYING POLYNUCLEOTIDES

• US 20080318802A1
• Filed: 04/28/2008
• Published: 12/25/2008
• Est. Priority Date: 02/10/2005
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Granted Patent: US 8,148,068 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 submixtues 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

1-21. -21. (canceled)

22. A method of identifying a polynucleotide selected from a mixture of tagged polynucleotides, the method comprising the steps of:

selecting one or more tagged polynucleotide from a mixture of tagged polynucleotides, said mixture comprising polynucleotides from a plurality of different polynucleotide samples wherein the polynucleotides in each of said different polynucleotide samples are tagged with a unique oligonucleotide tag comprising one or more words; and

determining the nucleotide sequence of at least one word of the unique oligonucleotide tag on said selected one or more tagged polynucleotide to identify from which of said plurality of different polynucleotide samples each of said selected one or more tagged polynucleotide is derived.

23. The method of claim 22, wherein said selecting step selects tagged polynucleotides from said mixture of tagged polynucleotides having a genetic variation.

24. The method of claim 22, wherein said selecting step selects tagged polynucleotides from said mixture of tagged polynucleotides having a particular nucleic acid sequence at one or more polymorphic locus.

25. The method of claim 22, wherein said selecting is based on one or more of:

specific hybridization and differential duplex stability, template-driven ligation, template-driven strand extension, and exonuclease digestion.

26. The method of claim 22, wherein said determining step comprises determining the nucleotide sequence of at least one word of the unique oligonucleotide tag on said selected one or more tagged polynucleotide in successive steps.

27. The method of claim 26, wherein each of said successive steps comprises contacting said selected one or more tagged polynucleotide under hybridizing conditions to a word-specific probe.

28. The method of claim 22 wherein each of said unique oligonucleotide tags comprises multiple words, said multiple words having an order, and wherein said determining step comprises determining the order of said words.

29. The method of claim 22, wherein said determining step comprises determining the nucleotide sequence of at least one word of the unique oligonucleotide tag on said selected one or more tagged polynucleotide simultaneously.

30. The method of claim 29, wherein said determining step comprises contacting said selected one or more tagged polynucleotide under hybridizing conditions to an addressable array comprising complements to each of said unique oligonucleotide tags.

31. The method of claim 30, wherein said addressable array is a microarray.

32. The method of claim 22, wherein said plurality of different polynucleotide samples are a plurality of different genomic samples.

33. The method of claim 22, wherein said polynucleotides in each of said plurality of different polynucleotide samples are polynucleotide fragments.

34. The method of claim 33, wherein said polynucleotide fragments are produced by restriction endonuclease digestion.

35. The method of claim 22, wherein each of said unique polynucleotide tags comprises from four to eight words.

36. The method of claim 22, wherein each of said words comprises from 2 to 8 nucleotides.

37. The method of claim 22, wherein each of said unique oligonucleotide tags is a ligation tag.

38. The method of claim 22, wherein each of said unique oligonucleotide tags includes a concatenate of words (W1, W2, W3, W4), wherein W1, W2, W3, and W4 are in any order, and W1 is selected from the group consisting of:

39. The method of claim 22, wherein each of said unique oligonucleotide tags includes a concatenate of words (W1, W2, W3, W4), wherein W1, W2, W3, and W4 are in any order, and W1 is selected from the group consisting of:

40. The method of claim 22, further comprising producing said mixture of tagged polynucleotides, said producing step comprising:

(a) providing;

(i) a mutually discriminable set containing a plurality of different words; and

(ii) said plurality of different polynucleotide samples;

(b) attaching one or more of said different words to the polynucleotides in each of said plurality of different polynucleotide samples to form tag-polynucleotide conjugates, wherein the polynucleotides in each of said different polynucleotide samples are tagged with a unique oligonucleotide tag comprising one or more word; and

(c) combining said tag-polynucleotide conjugates to produce said mixture of tagged polynucleotides.

41. The method of claim 22, further comprising producing said mixture of tagged polynucleotides, said producing step comprising:

(a) providing;

(i) a mutually discriminable set containing a plurality of different words; and

(ii) said plurality of different polynucleotide samples;

(b) attaching one or more of said different words to the polynucleotides from each sample in a first subset of said plurality of different polynucleotide samples to form tag-polynucleotide conjugates, wherein said polynucleotides in each sample of said first subset are thus tagged with a unique word or unique order of words;

(c) repeating step (b) for each subsequent subset of said plurality of different polynucleotide samples to form a base tier of tag-polynucleotide conjugates and recording which unique word or unique order of words is attached to the polynucleotides from each different polynucleotide sample in the base tier;

(d) combining tag-polynucleotide conjugates in each subset of the base tier to form a plurality of combined submixtures wherein the tag-polynucleotide conjugates from any particular polynucleotide sample in each combined submixture have an oligonucleotide tag that is different from that of any other poly nucleotide sample in the same combined submixture;

(e) attaching a different word to each different submixture of step (d) to form another tier of submixtures of tag-polynucleotide conjugates and recording which word is attached to each tag-polynucleotide conjugate in each submixture; and

(f) repeating steps (d) and (e) for the plurality of combined submixtures until only a single combined submixture is produced in step (d).