Labeled reactants and their uses

• US 8,906,614 B2
• Filed: 11/29/2012
• Issued: 12/09/2014
• Est. Priority Date: 03/13/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Wegener, Jeffrey, Korlach, Jonas
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/688,667
• Publication Number:
  US 20130183663A1
• Time in Patent Office: 740 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.33, 536/26.6
• US Class Current: 435/6.1
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Labeled reactant compositions, and particularly labeled nucleic acid reaction compositions, that include structural components that maintain potentially damaging labeling components sufficiently distal from the reactant portion of the molecule such that damaging effects of the label group on other reaction components, such as enzymes, are reduced, minimized and/or eliminated.

Claims

1. A method of monitoring an enzyme reaction, comprising:

providing a reaction mixture comprising the enzyme and at least a first reactant composition, the reactant composition comprising a compound having (i) a reactant component comprising a nucleotide, (ii) a fluorescent label component, and (iii) a linker component joining the reactant component to the label component, wherein the linker component is coupled to the reactant component through a phosphate group on the nucleotide, and the linker component maintains the label component at a functional distance away from the reactant component such that a reduction in the activity of the enzyme is reduced by at least 20%;

illuminating the reaction mixture to excite the fluorescent label component; and

detecting a fluorescent signal from the reaction mixture characteristic of the enzyme reaction.

2. The method of claim 1, wherein a reduction in the activity of the enzyme is reduced by at least 50%|.

3. The method of claim 1, wherein a reduction in the activity of the enzyme is reduced by at least 90%|.

4. The method of claim 1, wherein a reduction in the activity of the enzyme is a photo-induced decrease in enzyme activity.

5. The method of claim 1, wherein the linker component maintains the label component at a functional distance of at least 5 nm from the reactant component.

6. The method of claim 1, wherein the linker component maintains the label component at a functional distance of at least 10 nm from the reactant component.

7. The method of claim 1, wherein the linker component comprises groups selected from conjugated alkynes, conjugated alkenes, aryl alkynes, aryl amides, polyethylene glycol, oligopeptide, nucleic acids, PNA, and LNA.

8. The method of claim 1, wherein the linker component comprises an oligopeptide linker.

9. The method of claim 8, wherein the oligopeptide linker comprises oligoproline.

10. The method of claim 1, wherein the linker component comprises an oligonucleotide linker.

11. The method of claim 10, wherein the oligonucleotide linker comprises a double stranded oligonucleotide linker.

12. The method of claim 1, wherein the linker component comprises more than one ringed alkyl group.

13. The method of claim 12, wherein the more than one ringed alkyl group comprises more than one aromatic group.

14. The method of claim 13, wherein the one or more aromatic groups comprises at least one aryl alkyne group.

15. The method of claim 11, wherein the reactant component is coupled to a first strand of the double stranded oligonucleotide linker and the label component is coupled to a second strand of the oligonucleotide linker.

16. The method of claim 1, wherein the linker component comprises a PEG linker.

17. A method of nucleic acid sequencing, comprising:

providing a reaction mixture comprising a polymerase enzyme, a template nucleic acid having a sequence, and a reactant composition, the reactant composition comprising nucleotide analogs, each having (i) a reactant portion comprising a nucleotide, (ii) a fluorescent label portion, and (iii) a linker joining the reactant portion to the label portion, wherein the linker is coupled to the reactant portion through a phosphate group on the nucleotide, and the linker component maintains the label component at a functional distance away from the reactant component such that a reduction in the activity of the enzyme is reduced by at least 20%;

illuminating the reaction mixture to excite the fluorescent label portions; and

detecting fluorescent signals to determine nucleotide incorporation in real time, thereby obtaining information about the template sequence.

18. The method of claim 17, wherein a reduction in the activity of the enzyme is reduced by at least 50%|.

19. The method of claim 17, wherein a reduction in the activity of the enzyme is reduced by at least 50%|.

20. The method of claim 17, wherein a reduction in the activity of the enzyme is a photo-induced decrease in enzyme activity.

21. The method of claim 17, wherein the linker component comprises groups selected from conjugated alkynes, conjugated alkenes, aryl alkynes, aryl amides, polyethylene glycol, oligopeptide, nucleic acids, PNA, and LNA.

22. The method of claim 17, wherein the linker component comprises a PEG linker.

23. The method of claim 17, wherein the linker component maintains label component at a functional distance of at least 5 nm from the reactant component.

24. The method of claim 17, wherein the linker component maintains the label component at a functional distance of at least 10 nm from the reactant component.