Methods and compositions for incorporating nucleotides

Patent metadata

Field	Value
Publication number	US-10472382-B2
Application number	US-201816193639-A
Country	US
Kind code	B2
Filing date	Nov 16, 2018
Priority date	Mar 19, 2008
Publication date	Nov 12, 2019
Grant date	Nov 12, 2019

Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable Media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.

First claim

We claim:

1. A labeled reversible terminator comprising a nucleotide analogue of dUTP having a 3′-O group and a label, wherein the label is Rhodamine 6G and is attached to the nucleotide analogue of dUTP though a cleavable linker comprising a disulfide bond, and wherein a removable chemical moiety caps the 3′-O group, wherein said labeled reversible terminator is in a mixture in solution with an unlabeled reversible terminator comprising a nucleotide analogue of dUTP, wherein the concentration of the unlabeled nucleotide analogue of dUTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dUTP.

2. The labeled reversible terminator of claim 1 , wherein said mixture further comprises labeled and unlabeled reversible terminator nucleotide analogues of dCTP and dGTP.

3. The labeled reversible terminator of claim 2 , wherein said unlabeled nucleotide analogues are present at a concentration between 1 uM and 100 uM.

4. The labeled reversible terminator of claim 3 , wherein said labeled nucleotide analogues are present at a concentration between 1 nM and 1 uM.

5. A labeled reversible terminator comprising a nucleotide analogue of dGTP having a 3′-O group and a label, wherein the label is Cy5 and is attached to the nucleotide analogue of dGTP though a cleavable linker comprising a disulfide bond, and wherein a removable chemical moiety caps the 3′-O group, wherein said labeled reversible terminator is in a mixture in solution with an unlabeled reversible terminator comprising a nucleotide analogue of dGTP, wherein the concentration of the unlabeled nucleotide analogue of dGTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dGTP.

6. The labeled reversible terminator of claim 5 , wherein said mixture further comprises labeled and unlabeled reversible terminator nucleotide analogues of dCTP and dATP.

7. The labeled reversible terminator of claim 6 , wherein said unlabeled nucleotide analogues are present at a concentration between 1 uM and 100 uM.

8. The labeled reversible terminator of claim 7 , wherein said labeled nucleotide analogues are present at a concentration between 1 nM and 1 uM.

Assignees

• Qiagen Sciences Llc

Inventors

• Gordon Steven
• Olejnik Jerzy

Classifications

• G01N2021/6421

  Measuring at two or more wavelengths · CPC title

• G01N2021/6441

  with two or more labels · CPC title

• G01N2021/058

  Flat flow cell · CPC title

• C07H19/00 Primary

  Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides ; Anhydro-derivatives thereof · CPC title

• G01N2021/0346

  Capillary cells; Microcells · CPC title