Nucleotide analogs

• US 7,678,894 B2
• Filed: 05/18/2007
• Issued: 03/16/2010
• Est. Priority Date: 05/18/2007
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Siddiqi, Suhaib
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/804,591
• Publication Number:
  US 20080286837A1
• Time in Patent Office: 1,033 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/26.6, 536/25.3, 435/6, 435/91.1
• US Class Current: 536/23.1
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The disclosure provides nucleotide analogs and methods of their use. Analogs of the invention comprise a reporter molecule (label) attached via the N4, N6, O4, or O6 position of the nitrogenous base portion of the analog. In a preferred embodiment, nucleotide analogs of the invention comprise a label attached to the nitrogenous base portion of the analog via a cleavable linker at the N4, O4, N6 or O6 position.

Claims

1. A nucleotide analog, comprising:

A nitrogenous base;

A linker attached at the N4, N6, O4, or O6 of said nitrogeneous base; and

A detectable label attached to said nitrogenous base via said linker,

wherein said linker comprises a cleavable bond such that a native nucleotide monophosphate is regenerated upon incorporation of said analog into a nucleic acid duplex.

2. The nucleotide analog of claim 1, further comprising a mono-, di-, tri-, or bis-phosphate.

3. The nucleotide analog of claim 1, further comprising a ribose sugar or a deoxyribose sugar.

4. The nucleotide analog of claim 1, wherein said analog has the structure:

5. The nucleotide analog of claim 4, wherein X is an alkyl group or an aryl group.

6. The nucleotide analog of claim 4, wherein said alkyl group comprises between about 2 and about 10 atoms.

7. The nucleotide analog of claim 1, wherein said label is selected from cyanine-5, Cy-5, Cy-3, Joe, Rox, and TAMRA.

8. The nucleotide analog of claim 1, wherein said analog has the structure:

9. The nucleotide analog of claim 8, wherein X is (CH₂)_n, and wherein n is between about 2 and about 10.

10. The nucleotide analog of claim 8, wherein Y is a carbonyl.

11. The nucleotide analog of claim 8, wherein said label is an optically-detectable label.

12. The nucleotide analog of claim 11, wherein said detectable label is selected from cyanine-5, Cy-5, Cy-3, Joe, Rox, and TAMRA.

13. A method for sequencing a nucleic acid, the method comprising the steps of:

anchoring a nucleic acid duplex to a surface, the duplex comprising a template portion and a primer portion hybridized thereto;

Exposing said duplex to an analog of claim 4 or claim 10 in the presence of a polymerase capable of catalyzing addition of said analog to said primer portion in a template-dependent manner;

Removing unincorporated analog and polymerase;

Detecting incorporation of analog into said primer; and

Repeating said exposing, removing, and detecting steps at least once.

14. The method of claim 13, further comprising the step of cleaving detectable label from said analog after said detecting step.