Multiplexed analytical platform

• US 20050059065A1
• Filed: 09/02/2004
• Published: 03/17/2005
• Est. Priority Date: 09/09/2003
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Granted Patent: US 7,365,179 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The invention provides a system and reagents for carrying out multiplexed analytical measurements using the hybridization of oligonucleotide tags to addressable solid phase supports to obtain simultaneous readouts from hundreds to tens of thousands of reactions. In one aspect of the invention, analyte interaction moieties, such as oligonucleotide probes, are produced that are each labeled with a unique synthesis tag constructed from a set of two-nucleotide “words.” During or after an assay, the synthesis tags are converted to hybridization tags that are used in the readout step. Hybridization tags are constructed to maximize discrimination in the readout. Hybridization tags of the invention are preferably constructed from oligonucleotide “words” selected from a minimally cross-hybridizing set using combinatorial synthesis techniques. In a further aspect, discrimination in the readout is enhanced by employing hybridization tags that are constructed with words having the “comma-less” property and by minimizing or eliminating extraneous nucleic acids associated with the hybridization tag during a readout step.

Claims

1. A system for measuring one or more properties of each of a plurality of analytes, the system comprising:

a first set of analyte-interaction moieties capable of reacting with the plurality of analytes under assay reaction conditions, each analyte-interaction moiety having a synthesis tag;

conversion reagents for converting each synthesis tag into a hybridization tag and for releasing the hybridization tag from the analyte-interaction moieties; and

a solid phase detection array for detecting the released hybridization tags.

2. The system of claim 1 wherein said hybridization tag comprises one or more words selected from a minimally cross-hybridizing set, each word of the minimally cross-hybridizing set being an oligonucleotide having a length in the range of from four to nine nucleotides.

3. The system of claim 2 wherein said hybridization tag is comma-less.

4. The system of claim 3 wherein said analyte-interaction moiety is an oligonucleotide.

5. The system of claim 4 wherein said words of said hybridization tag each have a length in the range of from 4 to 5 nucleotides, wherein said words of said synthesis tag have a length of two nucleotides, and wherein said solid phase detection array is a microarray having from 100 to 100,000 hybridization sites.

6. A method of measuring one or more properties of each of a plurality of analytes in an assay reaction, the method comprising:

combining a first set of analyte-interaction moieties with the plurality of analytes under assay reaction conditions, each analyte-interaction moiety having a synthesis tag, each synthesis tag comprising a plurality of synthesis words, the plurality of synthesis words being a number in the range of from 4 to 32;

selecting products of the assay reaction, the products comprising a subset of analyte-interaction moieties;

converting the synthesis tags of the analyte-interaction moieties of the selected products into hybridization tags;

releasing the hybridization tags from the analyte-interaction moieties; and

applying the hybridization tags to a solid phase detection array to identify the analyte-reaction moieties that formed products.

7. The method of claim 6 wherein said step of converting includes:

(a) cleaving a synthesis word in each of said synthesis tags attached to said analyte-interaction moieties to form a reaction mixture, the cleaved synthesis word of each synthesis tag being converted to single-stranded form;

(b) separating the reaction mixture into a number of aliquots greater than or equal to said number of synthesis words;

(c) treating each aliquot to destroy every said synthesis tag, except for said synthesis tags having a cleaved synthesis word of a predetermined type, each different aliquot having a cleaved synthesis word of a different predetermined type;

(d) adding a hybridizaton tag corresponding to the synthesis word of the predetermined type in each of the aliquots; and

(e) repeating steps (a) through (d) until said synthesis tag is converted into said hybridization tag.

8. A composition of matter comprising a plurality of hybridization tags, the hybridization tags the following structure:

W₁W₂ . . . W_n

wherein each W_i for i=1, 2, . . . n is selected from a group of oligonucleotides having nucleotide sequences;

9. A solid phase support having attached a plurality of tag complements, the solid phase support comprising:

a plurality of addressable sites, each addressable site having substantially identical copies of only one tag complement, the tag complement having the structure;

W₁W₂ . . . W_n

wherein each W_i for i=1, 2, . . . n is selected from a group of oligonucleotides having nucleotide sequences;

AICATGTCTA GAACTTGTCA CTTGTACAGA TCITTCAGAA

or

CTGTACAAGT TCTGAACIAT AGACATICTT GACAAGTTCT.

10. A microarray comprising:

a planar non-porous solid phase support having a surface; and

a plurality of different oligonucleotides attached to the surface, wherein each of the different oligonucleotides is attached to the surface in a different predefined region, and wherein the plurality of different oligonucleotides comprises a set of comma-less tags.

11. A method of producing a plurality of hybridization tags, the method comprising the steps of:

producing on a solid phase support a plurality of synthesis tags, each synthesis tag comprising a plurality of two-nucleotide words;

copying the plurality of synthesis tags; and

converting each two-nucleotide word of each of the synthesis tags into a corresponding hybridization word of each of the hybridization tags.

12. The method of claim 11 wherein said corresponding hybridization word has a length in the range of from four to five nucleotides.