Methods and compositions for incorporating nucleotides

Patent metadata

Field	Value
Publication number	US-11001887-B2
Application number	US-201916430064-A
Country	US
Kind code	B2
Filing date	Jun 3, 2019
Priority date	Mar 19, 2008
Publication date	May 11, 2021
Grant date	May 11, 2021

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

The invention claimed is:

1. A method of incorporating labeled nucleotides into nucleic acid, comprising:
a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, and a mixture of labeled and non-labeled reversible nucleotide analogue terminators, said labeled reversible nucleotide analogue terminators comprising a label linked to a nucleotide analogue, said label producing color, wherein said labeled reversible nucleotide analogues are labeled with at least one type of fluorescent dye; wherein each reversible terminator contains a reversibly removable chemical moiety capping the 3′-OH group;
b) incorporating a first labeled nucleotide analogue with said polymerase;
c) detecting color of said at least one type of fluorescent dye of the incorporated nucleotide analogue in two channels;
d) removing i) said label of the incorporated nucleotide analogue and ii) said reversibly removable chemical moiety capping the 3′-OH group, with said cleaving agent; and
e) incorporating a second nucleotide analogue.

2. The method of claim 1 , wherein said label and said chemical moiety capping the 3′-OH group are removed at the same time.

3. The method of claim 1 , wherein said at least one type of fluorescent dye is visible in the yellow channel and the green channel.

4. The method of claim 1 , wherein said at least one type of fluorescent dye is visible in the red channel and the green channel.

5. The method of claim 1 , wherein said at least one type of fluorescent dye is visible in the blue channel and the red channel.

6. The method of claim 1 , wherein said non-labeled nucleotides are employed in ratios between 10:1 and 100:1 relative to the labeled nucleotides.

7. The method of claim 1 , wherein said mixture of labeled and non-labeled reversible nucleotide analogues is reversibly terminated by a disulfide bond at the 3′ position.

8. The method of claim 1 , wherein said mixture of labeled and non-labeled reversible terminators is reversibly terminated by an azidomethyl group at the 3′ position.

Assignees

• Qiagen Sciences Llc

Inventors

• Gordon Steven
• Olejnik Jerzy

Classifications

• G01N21/6428 Primary

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N2021/6441

  with two or more labels · CPC title

• G01N2021/6421

  Measuring at two or more wavelengths · CPC title

• G01N21/645

  Specially adapted constructive features of fluorimeters · CPC title

• G01N2021/6419

  Excitation at two or more wavelengths · CPC title