Engineered fluorescent dye labeled nucleotide analogs for DNA sequencing

• US 8,133,702 B2
• Filed: 05/25/2011
• Issued: 03/13/2012
• Est. Priority Date: 03/26/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Shen, Gene, Peluso, Paul, Bibillo, Arkadusz
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/115,284
• Publication Number:
  US 20120028248A1
• Time in Patent Office: 293 Days
• Field of Search: 435/91.1, 435/6.1, 536/23.1, 536/24.3
• US Class Current: 435/91.1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom

Abstract

Engineered nucleotide compositions, having polymerase interacting components that improve the interactivity of the polymerase and the nucleotide, particularly for nucleic acid sequencing applications. Compositions include the interactive polymerases along with the nucleotide analogs. Kits, methods and systems are provided for analysis of nucleic acid synthesis reactions.

Claims

1. A method of monitoring polymerase mediated, template dependent primer extension, comprising:

providing a polymerase template primer complex;

contacting the complex with a nucleotide composition, comprising the structure;

N—

Y—

F

wherein N is a nucleoside polyphosphate or analog thereof;

F is a detectable label moiety; and

Y comprises a polymerase interacting linkage complex comprising a polymerase interacting group positioned from 20 to 40 angstroms from a nucleoside portion of the nucleoside polyphosphate; and

monitoring reaction of the nucleotide composition with the complex in a primer extension reaction by detecting a signal from the detectable label.

2. The method of claim 1, wherein the nucleotide composition is selected from the group:

3. The method of claim 2, wherein R₁ through R₁₁ are independently selected from H, O, OH, guanidinium, amine, ammonium, iminium, oxonium, carboxyl, phosphate, phosphonate, thio, sulfate, sulfonate, thioate, and phosphothioate.

4. The method of claim 2, wherein the polymerase interacting linkage complex comprises one or more hydrophobic groups selected from phenyl, biphenyl, triphenyl and coumarin derivative groups.

5. The method of claim 4, wherein the one or more hydrophobic groups is selected from a chromene, 2-chromanone, 2H-chromene, or 2H-chromen-2-none group.

6. The method of claim 1, wherein the complex is immobilized upon a solid support.

7. The method of claim 1, wherein the polymerase interacting linkage complex (Y) comprises the structure:

-L₁-P-L₂-

where L₁ is a first linker group, P is a polymerase interacting group, and L₂ is a second linker group.

8. The method of claim 1, wherein the polymerase interacting linkage complex (Y) comprises the structure:

9. The nucleotide composition of claim 1, wherein the polymerase interacting component comprises a hydrophobic group having one or more phenyl groups.