Thiotriphosphate nucleotide dye terminators

• US 20060281100A1
• Filed: 06/14/2005
• Published: 12/14/2006
• Est. Priority Date: 06/14/2005
• Status: Abandoned Application

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Shen, Gene G.-Y., Michael, Josephine M.
• Application Number:
  US11/153,715
• Publication Number:
  US 20060281100A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Fluorescent reporter compounds having a chain terminating (thio)triphosphate nucleotide derivative, a fluorescent dye, and a linker of sufficient length to connect the nucleotide derivative to the fluorescent dye are provided. The fluorescent reporter compounds are used in DNA sequencing reactions and are substantially inactive toward exonuclease digestion.

Claims

1. A compound of the formula:

wherein

Z is a (thio)triphosphate nucleotide derivative;

D is a fluorescent dye; and

L is a linker of sufficient length to connect the (thio)triphosphate nucleotide to the fluorescent dye, such that the fluorescent dye does not significantly interfere with the overall binding and recognition of the nucleotide derivative by a nucleic acid replication enzyme.

2. A compound according to claim 1 wherein Z is an α

-(thio)triphosphate dideoxynucleotide.

3. A compound according to claim 1 wherein D is a dye having a near infrared fluorescence.

4. A compound according to claim 3 wherein D is a fluorescent cyanine dye.

5. A compound according to claim 4 wherein D is a fluorescent cyanine dye of the formula:

wherein

Z is defined as in claim 1;

A and B are each independently ring structures having sufficient atoms to form a cyanine nuclei;

n is 0, 2, 3, or 4;

p is 0 or 1;

R₁ is selected from the group consisting of C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogen, and hydroxy with the proviso that R₁ is not present when n is 0;

R₂ is selected from the group consisting of C₁-C₂₀ alkyl and hydrogen, wherein the C₁-C₂₀ alkyl is unsubstituted or substituted with C₁-C₄ alkoxy, C₁-C₄ alkoxycarbonyl, C₁-C₄ alkyl, amine, amide, C₁-C₄ carboxy, C₁-C₄ carboxylate, halogen, hydroxy, and sulfonate;

R₃ is selected from the group consisting of hydrogen, halogen, OR₇, SR₇, NR₇R₈, C₁-C₆ alkyl, C₁-C₆ alkylene, C₃-C₆ cycloalkyl, C₃-C₆ cycloheteroalkyl, C₃-C₆ cycloalkylene, C₃-C₆ cycloheteroalkylene, phenyl, biaryl, heteroaryl, or heterobiaryl, wherein the C₁-C₆ alkyl, C₁-C₆ alkylene, C₃-C₆ cycloalkyl, C₃-C₆ cycloheteroalkyl, C₃-C₆ cycloalkylene, C₃-C₆ cycloheteroalkylene, phenyl, biaryl, heteroaryl and heterobiaryl groups are unsubstituted or substituted with C₁-C₄ alkyl, C₁-C₄ haloalkyl, halogen or hydroxy;

R₄ and R₅ are each independently selected from the group consisting of C₁-C₄ alkoxy, C₁-C₄ alkoxycarbonyl, C₁-C₄ alkyl, amine, amide, C₁-C₄ carboxy, C₁-C₄ carboxylate, halogen, hydroxy, and sulfonate;

R₇ and R₈ are each independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, phenyl, biaryl, heteroaryl, and heterobiaryl, wherein the C₁-C₆ alkyl, C₁-C₆ cycloalkyl, phenyl, biaryl, heteroaryl, and heterobiaryl groups may be substituted with halogen, OH, C₁-C₄ alkyl, and C₁-C₄ haloalkyl, or when R₃ represents NR₇R₈, R₇ and R₈ may be taken together to form an optionally substituted

C₃-C₆ aliphatic or C₃-C₆ aromatic heterocyclic ring.

6. A compound according to claim 1 wherein Z is selected from the group consisting of:

7. A compound according to claim 1 wherein L is a linker of the formula:

wherein m is an integer from 1 to 20.

8. A compound according to claim 1 of the formula:

wherein

Z is defined as in claim 1;

m is an integer from 1 to 20.

p is 0 or 1;

and

R₂ is selected from the group consisting of C₁-C₂₀ alkyl and hydrogen, wherein the C₁-C₂₀ alkyl is unsubstituted or substituted with C₁-C₄ alkoxy, C₁-C₄ alkoxycarbonyl, C₁-C₄ alkyl, amine, amide, C₁-C₄ carboxy, C₁-C₄ carboxylate, halogen, hydroxy, and sulfonate.

9. A compound according to claim 1 of the formula:

wherein

Z is defined as in claim 1;

m is an integer from 1 to 20;

p is 0 or 1;

and

R₂ is selected from the group consisting of C₁-C₂₀ alkyl and hydrogen, wherein the C₁-C₂₀ alkyl is unsubstituted or substituted with C₁-C₄ alkoxy, C₁-C₄ alkoxycarbonyl, C₁-C₄ alkyl, amine, amide, C₁-C₄ carboxy, C₁-C₄ carboxylate, halogen, hydroxy, and sulfonate.

10. A compound according to claim 1 of the formula:

wherein

X are the atoms sufficient to make up a heterocyclic base; and

Cy is a fluorescent dye.

11. A compound according to claim 10 wherein Cy is a cyanine dye.

12. A method of nucleic acid sequence analysis comprising:

reacting a compound according to claim 1 with a first nucleic acid sequence to produce a second nucleic acid sequence labeled with the fluorescent reporter-labeled compound; and

detecting the reporter on the second nucleic acid sequence.

13. A method for determining the base sequence of a target DNA comprising:

providing a mixture of compounds according to claim 1, the mixture of compounds corresponding to each of the four DNA bases;

providing a DNA template corresponding to the target DNA;

reacting the DNA template with a DNA primer;

extending the DNA template and DNA primer with a replication enzyme, a mixture of DNA nucleotides, and the mixture of compounds to produce a plurality of DNA fragments, each DNA fragment having a fluorescent reporter labeled compound attached to the 3′

-terminal residue of the DNA fragment;

separating the plurality of fluorescent reporter labeled DNA fragments; and

detecting the fluorescent reporter on each separated fluorescent reporter labeled DNA fragment to determine the base sequence of the target DNA.

14. A method of nucleic acid sequence analysis comprising:

providing a template nucleic acid;

providing one or more fluorescent reporter labeled (thio)triphosphate nucleotide compounds;

reacting a nucleotide primer with a replication enzyme, a mixture of nucleotide precursors, and the one or more fluorescent reporter labeled (thio)triphosphate nucleotide compounds to extend the nucleotide primer and produce a plurality of nucleotide fragments having a fluorescent reporter labeled (thio)triphosphate nucleotide compound attached to the 3′

-terminal residue of each nucleotide fragment;

separating the plurality of nucleotide fragments; and

detecting the fluorescent reporter for each separated fluorescent reporter labeled nucleotide fragment.

15. A method according to claim 14 wherein at least one of the fluorescent reporter labeled (thio)triphosphate nucleotide compounds is labeled with a cyanine dye.

16. A method according to claim 14 wherein at least one of the fluorescent reporter labeled (thio)triphosphate nucleotide compounds is labeled with a dye having a near infrared fluorescence.

17. A method according to claim 14 wherein four different fluorescent reporter labeled (thio)triphosphate nucleotide compounds are provided, each fluorescent reporter labeled chain terminating (thio)triphosphate nucleotide compound being labeled with a dye having a near infrared fluorescence.

18. A method according to claim 17 wherein each of the four different fluorescent reporter labeled (thio)triphosphate nucleotide compounds is labeled with a cyanine dye.