Enzyme-linked nucleotides

Patent metadata

Field	Value
Publication number	US-10767221-B2
Application number	US-201314776551-A
Country	US
Kind code	B2
Filing date	Mar 15, 2013
Priority date	Mar 15, 2013
Publication date	Sep 8, 2020
Grant date	Sep 8, 2020

Abstract

Presented herein are polymerase-linked nucleotides for improved distinguishing nucleotide sequences for different nucleic acid molecules. Also presented are methods and systems using the polymerase-linked nucleotides for improved distinguishing nucleotide sequences for different nucleic acid molecules.

First claim

What is claimed is:

1. A modified DNA polymerase comprising:
a) a point mutation wherein a surface-exposed amino acid residue is replaced with a cysteine residue;
b) a flexible linker having a first end and second end, the first end covalently attached to the cysteine residue;
c) a nucleotide attached to the second end of the flexible linker, wherein the flexible linker attachment to the nucleotide allows, the nucleotide to interact at a catalytically active region of said modified DNA polymerase and incorporate the nucleotide to the 3′ end of a polynucleotide in the presence of a template nucleic acid sequence thereby producing a detectable increase in dwell time at the site of incorporation; and
c) an optically detectable label comprising a fluorophore attached to the modified DNA polymerase.

2. The modified DNA polymerase of claim 1 , wherein the linker attachment to the nucleotide occurs at the 5′ gamma phosphate of the nucleotide.

3. The modified DNA polymerase of claim 1 , wherein said nucleotide comprise one of the group of nucleotides consisting of: dCTP, dATP, dGTP, dTTP, and dUTP.

4. The modified DNA polymerase of claim 1 , wherein said nucleotide comprises polymeric phosphate comprising 3, 4, 5, 6 or greater than 6 phosphates.

5. The modified DNA polymerase of claim 4 , wherein said polymeric phosphate comprises hexaphosphate.

6. The modified DNA polymerase of claim 1 , wherein said flexible linker is of a length so as to create a relative concentration of said nucleotide in solution of greater than 1 μM, 10 μM, 100 μM, or greater than 1 mM.

7. The modified DNA polymerase of claim 1 , said flexible linker having a length of greater than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or greater than 200 Angstrom.

8. The modified DNA polymerase of claim 1 , said flexible linker having a length of less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 or less than 50 Angstrom.

Assignees

• Illumina Inc

Inventors

• Gloeckner Christian
• Kellinger Matthew William
• Pickering Lea

Classifications

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title

• C12N9/1276

  RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase · CPC title

• A61K47/59

  obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• A61K47/64

  Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65) · CPC title