Multiplex analytical platform using molecular tags

• US 20030207300A1
• Filed: 01/07/2003
• Published: 11/06/2003
• Est. Priority Date: 04/28/2000
• Status: Abandoned Application

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat S., Macevicz, Stephen C., Matray, Tracy J.
• Application Number:
  US10/338,729
• Publication Number:
  US 20030207300A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2563/167 Mass label

Abstract

Compositions and methods are disclosed for detecting multiple target analytes, particularly polynucleotide target analytes. In accordance with one aspect of the invention, a template-dependent extension reaction is performed to generate detection probes, such that each detection probe has (i) at least one molecular tag attached by a cleavable linkage and (ii) either a capture moiety or a cleavage-inducing moiety attached. The template-dependent extension reaction may be carried out directly on a polynucleotide analyte to generate molecular tags, wherein the polynucleotide analyte serves as a template in the template-dependent extension reaction, or it may be carried out indirectly on an oligonucleotide label that, in turn, is attached to a binding moiety specific for an analyte of interest. In either case, a plurality of molecular tags are generated, after which they are separated and identified to determine the presence or absence or the quantity of the target analytes in a sample.

Claims

1. A method of generating molecular tags indicative of a plurality of polynucleotides in a sample, the method comprising the steps of:

extending a primer annealed to each polynucleotide to form a detection probe under conditions that permit dissociation of detection probes from the polynucleotides after extension, each detection probe having a molecular tag and either a sensitizer with an effective proximity or a capture moiety, the molecular tag being attached by a cleavable linkage and being within the effective proximity of the sensitizer upon dissociation of the detection probe from the polynucleotide whenever the detection probe has a sensitizer attached, and the molecular tag being selected from a plurality of molecular tags such that each molecular tag of the plurality has one or more physical and/or optical characteristics distinct from those of the other molecular tags of the plurality so that each molecular tag forms a distinguishable peak upon cleavage and separation based on such one or more physical and/or optical characteristics;

generating detectable amounts of detection probes in said step of extending;

activating the sensitizers to generate an active species so that the cleavable linkages are cleaved and the molecular tags are released; and

separating and identifying the released molecular tags to determine the plurality of polynucleotides in the sample.

2. The method of claim 1 wherein said step of extending includes extending with a DNA polymerase said primer by a terminator, the terminator having said sensitizer attached or said capture moiety attached.

3. The method of claim 2 wherein said terminator has said capture moiety attached and wherein after said step of generating detectable amounts of said detection probes, a further step of capturing each of said detection probes by a complementary moiety of said capture moiety, the complementary moiety being attached to a photosensitizer bead.

4. The method of claim 3 wherein said capture moiety is biotin and said complementary moiety is avidin or streptavidin.

5. The method according to claims 1, 2, 3, or 4 wherein said step of separating is electrophoretically separating or chromatographically separating, and wherein said molecular tag has a molecular weight in the range of from 100 to 2500 daltons.

6. The method of claim 5 wherein said molecular tags consist of said plurality of molecular tags selected from a group defined by the formula:

—

L—

(M,D)

wherein;

L is a cleavable linkage;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

7. The method of claim 6 wherein said plurality is in the range of from 2 to 100 and wherein D is a fluorescent label.

8. A composition of matter defined by the formula:

9. A composition of matter defined by the formula:

10. The composition of claim 9 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles having from 6 to 100 atoms, not including hydrogen, selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

11. A composition of matter comprising:

one or more photosensitizer beads having a complementary moiety attached, the complementary moiety being capable of capturing a capture moiety; and

one or more oligonucleotides each having attached a capture moiety and a molecular tag, the molecular tag being attached by a cleavable linkage, and each molecular tag being selected from a plurality of molecular tags such that each molecular tag of the plurality has one or more physical and/or optical characteristics distinct from those of the other molecular tags of the plurality so that each molecular tag forms a distinguishable peak upon cleavage and separation based on such one or more physical and/or optical characteristics;

wherein each of the one or more oligonucleotides are attached to the one or more photosensitizer beads by specific binding of the capture moiety to the complementary moiety.

12. The composition of claim 11 wherein said separation is electrophoretic separation or chromatographic separation, and wherein said molecular tag has a molecular weight in the range of from 100 to 2500 daltons.

13. The composition of claim 12 wherein each of said molecular tags attached to said one or more oligonucleotides is selected from a group defined by the formula:

—

L—

(M,D)

wherein;

L is a cleavable linkage;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

14. The composition of claim 13 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

15. The composition of claim 14 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

16. The composition of claim 15 wherein D is a fluorescein.

17. The composition of claim 16 wherein said fluorescein is selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.

18. The composition of claim 13 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

19. The composition in accordance with claims 11, 12, 13, 14, 15, 16, 17, or 18 wherein said plurality of molecular tags is in the range of from 2 to 100, and wherein said separation is electrophoretic separation.

20. The composition of claim 19 wherein said plurality of molecular tags is in the range of from 3 to 50.