Fluorescent nucleotide analogs and uses therefor

• US 7,405,281 B2
• Filed: 09/29/2005
• Issued: 07/29/2008
• Est. Priority Date: 09/29/2005
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Wegener, Jeffrey, Xu, Yue, Bibillo, Arek
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/241,809
• Publication Number:
  US 20070072196A1
• Time in Patent Office: 1,034 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/26.6, 536/4.1, 435/6, 435/91.1
• US Class Current: 536/4.1
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

Labeled nucleotide analogs used in place of naturally occurring nucleoside triphosphates or other analogs in template directed nucleic acid synthesis reactions and other nucleic acid reactions, and various analyses based thereon, including DNA sequencing, single base identification, hybridization assays and others.

Claims

1. A composition, comprising a labeled nucleotide analog, comprising the formula:

2. The composition of claim 1, wherein at least one of R₂, R₃ is not O.

3. The composition of claim 1, wherein R₄ comprises:

4. The composition of claim 3, wherein at least one of R₂, R₃, R₉ and R₁₀ is not O.

5. The composition of claim 1, wherein 4 is

6. The composition of claim 5, wherein at least one of R₂, R₃, R₉, R₁₀ and R₁₂ is not O.

7. The composition of claim 1, wherein at least one of R₂, R₃ and R₄ is CR′

R″

, where R′ and

R″

are independently selected from H, F, Cl, OH, NH₂, alkyl, aminoalkyl, alkenyl, alkynyl, aryl, and heterocycle.

9. The composition of claim 1, wherein R₁ is O, at least one of R₂ and R₃ is CH₂, at least one of R₂ and R₃ is O, and R₄ is selected from O and NH.

10. The composition of claim 9, wherein R₅ is BH₃.

11. The composition of claim 1, wherein R₁, R₂, R₃, R₄, R₆, R₇ and R₈ are O, and R₅ is BH₃.

12. The composition of claim 3, wherein R₁, R₅, R₆, R₇, R₈, R₉, R₁₁ are O, and at least one of R₂ and R₃ is CH₂ and R₁₀ is selected from NH and O.

13. The composition of claim 1, wherein B is selected from adenine, guanine, cytidine, thymine, uracil, and inosine.

14. The composition of claim 1, wherein S is selected from D-ribosyl, D-deoxyribosyl and D-dideoxyribosyl.

15. The composition of claim 1, wherein L comprises a fluorescent label.

16. The composition of claim 1, wherein L comprises at least one component of an energy transfer dye complex.

17. A method of determining an identity and relative position of a nucleotide in a template nucleic acid sequence, comprising:

providing the template nucleic acid sequence complexed with a polymerase enzyme capable of template dependent synthesis of a complementary nascent sequence, in a first complex;

contacting the first complex with the compound of claim 1, wherein the compound is complementary to a known nucleotide; and

detecting whether the compound is incorporated into the nascent strand, incorporation of the compound being indicative that the complementary nucleotide is in a position in the template nucleic acid that is being processed by the polymerase enzyme.

18. A method of determining an identity and relative position of a nucleotide in a template nucleic acid sequence, comprising:

providing the template nucleic acid sequence complexed with a polymerase enzyme capable of template dependent synthesis of a complementary nascent sequence, in a first complex;

contacting the first complex with the compound of claim 1, wherein the compound is complementary to a known nucleotide, and wherein the contacting step comprises contacting the first complex with a plurality of different compounds each comprising the compound of claim 1, wherein each different compound comprises a different base selected from A, T, G and C, and an individually detectable label.

19. A kit, comprising at least four differently labeled nucleotide analogs each comprising the structure of the composition of claim 1, a polymerase enzyme source, and instructions for using the composition with the polymerase enzyme in template directed nucleic acid synthesis.

8. A composition, comprising a labeled nucleotide analog, comprising the formula: