Methods and compositions for incorporating nucleotides

Patent metadata

Field	Value
Publication number	US-10961574-B2
Application number	US-201916674341-A
Country	US
Kind code	B2
Filing date	Nov 5, 2019
Priority date	Mar 19, 2008
Publication date	Mar 30, 2021
Grant date	Mar 30, 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

We claim:

1. A method for determining an identity of a nucleotide at a position, comprising: a) providing nucleic acid, polymerase, and a mixture of labeled and non-labeled reversible terminators, said labeled reversible terminators comprising a label linked to a nucleotide analogue, said label producing color, wherein the label is Rhodamine 6G and is attached to the nucleotide analogue of dUTP though a cleavable linker 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; b) incorporating, with said polymerase, a first nucleotide analogue from said mixture into said nucleic acid at a position; and c) detecting the label of said incorporated first nucleotide analogue.

2. The method of claim 1 , wherein said detecting is done with a device.

3. The method of claim 2 , wherein said device measures color in more than one detector channel.

4. The method of claim 3 , wherein said color detected in two channels.

5. The method of claim 1 , wherein said cleavable linker comprises a disulfide bond.

6. The method of claim 1 , further comprising d) determining the identity of the incorporated nucleotide at said position.

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

8. The method of claim 7 , wherein said unlabeled nucleotide analogues are present at a concentration between 1 uM and 100 uM.

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

10. A method for determining an identity of a nucleotide at a position, comprising: a) providing nucleic acid, polymerase, and a mixture of labeled and non-labeled reversible terminators, said labeled reversible terminators comprising a label linked to a nucleotide analogue, said label producing color, wherein the label is Cy5 and is attached to the nucleotide analogue of dGTP though a cleavable linker 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; b) incorporating, with said polymerase, a first nucleotide analogue from said mixture into said nucleic acid at a position; and c) detecting the label of said incorporated first nucleotide analogue.

11. The method of claim 10 , wherein said detecting is done with a device.

12. The method of claim 11 , wherein said device-measures color in more than one detector channel.

13. The method of claim 12 , wherein said color detected in two channels.

14. The method of claim 10 , wherein said cleavable linker comprises a disulfide bond.

15. The method of claim 10 , further comprising d) determining the identity of the incorporated nucleotide at said position.

16. The method of claim 10 , wherein said mixture further comprises labeled and unlabeled reversible terminator nucleotide analogues of dCTP and dATP.

17. The method of claim 16 , wherein said unlabeled nucleotide analogues are present at a concentration between 1 uM and 100 uM.

18. The method of claim 17 , wherein said labeled nucleotide analogues are present at a concentration between 1 nM and 1 uM.

19. A method for determining an identity of a nucleotide at a position, comprising: a) providing nucleic acid, polymerase, and a mixture of labeled and non-labeled reversible terminators, said labeled reversible terminators comprising a label linked to a nucleotide analogue, said label producing color, wherein the label is ROX and is attached to the nucleotide analogue of dATP though a cleavable linker 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 dATP, wherein the concentration of the unlabeled nucleotide analogue of dATP is present in said solution at a higher concentration than the labeled nucleotide analogue of dATP; b) incorporating, with said polymerase, a first nucleotide analogue from said mixture into said nucleic acid at a position; and c) detecting the label of said incorporated first nucleotide analogue.

20. The method of claim 19 , wherein said detecting is done with a device.

21. The method of claim 20 , wherein said device-measures color in more than one detector channel.

22. The method of claim 21 , wherein said color detected in two channels.

23. The method of claim 19 , wherein said cleavable linker comprises a disulfide bond.

24. The method of claim 19 , further comprising d) determining the identity of the incorporated nucleotide at said position.

25. The method of claim 19 , wherein said mixture further comprises labeled and unlabeled reversible terminator nucleotide analogues of dCTP and dGTP.

26. The method of claim 25 , wherein said unlabeled nucleotide analogues are present at a concentration between 1 uM and 100 uM.

27. The method of claim 26 , 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

• 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

• G01N2021/6419

  Excitation at two or more wavelengths · CPC title

• G01N2021/6421

  Measuring at two or more wavelengths · CPC title

• G01N2021/6471

  Special filters, filter wheel · CPC title

• G01N21/05

  Flow-through cuvettes (G01N21/09 takes precedence; handling fluid samples G01N1/10) · CPC title