Labeled enzyme compositions, methods and systems

Patent metadata

Field	Value
Publication number	US-9567629-B2
Application number	US-201414284187-A
Country	US
Kind code	B2
Filing date	May 21, 2014
Priority date	Mar 27, 2009
Publication date	Feb 14, 2017
Grant date	Feb 14, 2017

Abstract

Disclosed herein are conjugates comprising a biomolecule linked to a label that have biological activity and are useful in a wide variety of biological applications. For example, provided herein are labeled polymerase conjugates including a polymerase linked to one or more labels, wherein the conjugate has polymerase activity. Such conjugates can exhibit enhanced biological activity and/or superior detectability as compared to conventional labeled polymerases. Also disclosed herein are improved methods for preparing such conjugates, and methods and systems for using such conjugates in biological applications such as nucleotide incorporation, primer extension and single molecule sequencing.

First claim

What is claimed is:

1. A method for generating a signal, comprising: contacting a labeled polymerase conjugate with a labeled nucleotide under conditions where the labeled polymerase conjugate catalyzes the incorporation of the labeled nucleotide into a nucleic acid, wherein the labeled polymerase conjugate includes a polymerase linked to at least three optically-detectable dye labels, wherein the at least three optically-detectable dye labels are linked to one or more biotin acceptor peptide sequence of the polymerase, and wherein the polymerase has nucleotide incorporation activity, and wherein the labeled polymerase conjugate emits upon continuous excitation a signal having a total photon count of at least 10 6 photons before irreversibly photo-bleaching.

2. The method of claim 1 , further comprising detecting the signal.

3. The method of claim 1 , further comprising analyzing the signal to identify the nucleotide that is incorporated.

4. The method of claim 1 , wherein at least one of the three optically-detectable dye labels generates the signal.

5. The method of claim 1 , wherein the labeled polymerase conjugate has activity that is at least about 80% relative to the polymerase activity of the unconjugated polymerase.

6. The method of claim 1 , wherein the labeled polymerase conjugate comprises three, four, five, six, or seven optically-detectable dye labels.

7. The method of claim 1 , wherein the at least three optically-detectable dye labels are attached to different biotin acceptor peptide sequences of the polymerase.

8. The method of claim 1 , wherein the at least three optically-detectable dye labels are attached to a single biotin acceptor peptide sequence of the polymerase.

9. The method of claim 1 , wherein the labeled polymerase conjugate emits upon continuous excitation a total photon count of at least 10 8 photons before irreversibly photobleaching.

10. The method of claim 1 , wherein the labeled polymerase conjugate, further comprises at least one biotin moiety linked to the biotin acceptor peptide sequence.

11. The method of claim 10 , wherein the biotin acceptor peptide sequence comprises the amino acid sequence of SEQ ID NO:10.

12. The method of claim 10 , wherein the biotin moiety is linked to an avidin moiety.

13. The method of claim 12 , wherein the avidin moiety is linked to the at least three optically-detectable dye labels.

14. The method of claim 1 , wherein at least one of the three labels is positioned on the polymerase to undergo energy transfer with a labeled nucleotide bound to the nucleotide binding site of the labeled polymerase conjugate.

15. The method of claim 14 , wherein the energy transfer comprises FRET.

16. The method of claim 1 , wherein the labeled nucleotide comprises three, four, five, six, seven, eight, nine or ten phosphorus atoms.

17. The method of claim 16 , wherein the label of the labeled nucleotide is linked to the terminal phosphorus atom.

Assignees

• Life Technologies Corp

Inventors

• Nikiforov Theo

Classifications

• C07H19/20 Primary

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12Y207/07

  Nucleotidyltransferases (2.7.7) · CPC title

• G01N2021/6432

  Quenching · CPC title

• G01N33/582

  with fluorescent label · CPC title