Methods and compositions for incorporating nucleotides

• US 9,644,237 B2
• Filed: 05/02/2016
• Issued: 05/09/2017
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: Intelligent Bio-Systems Incorporated. (Qiagen NV)
• Inventors: Gordon, Steven, Olejnik, Jerzy
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/144,079
• Publication Number:
  US 20160348165A1
• Time in Patent Office: 372 Days
• Field of Search: 536 41, 536 261
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 2021/058 Flat flow cell
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/6428 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 Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G01N 2201/0627 : Use of several LED's for spectral resolution
• CPC Class Codes: G01N 2201/12 : Circuits of general importance; Signal processing
• CPC Class Codes: G16B 40/10 : Signal processing, e.g. from mass spectrometry [MS] or from PCR

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.

Claims

1. A composition comprising a mixture of labeled and unlabeled reversible terminators in solution, said terminators comprising nucleotide analogues of dCTP, dGTP and dATP, wherein the concentration of the unlabeled nucleotide analogue of dCTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dCTP, 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, 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, and wherein each labeled and unlabeled nucleotide analogue contains a removable chemical moiety capping the 3′

-OH group.

2. The composition of claim 1, wherein said terminators further comprise nucleotide analogues of dTTP, wherein the concentration of the unlabeled nucleotide analogue of dTTP is present in said solution at a higher concentration than the labeled nucleotide analogue of dTTP, wherein each labeled and unlabeled nucleotide analogue of dTTP contains a removable chemical moiety capping the 3′

-OH group.

3. The composition of claim 1, wherein said unlabeled reversible terminators are present at a concentration between 1 uM and 100 uM.

4. The composition of claim 1, wherein said first plurality of nucleotide analogues is present at a concentration between 1 nM and 1 uM.

5. The composition of claim 1, wherein the labeled reversible terminators comprise a label selected from the group consisting of BODIPY, Rhodamine, Carboxyrhodamine, and Cyanine.

6. The composition of claim 1, wherein said removable chemical moiety comprises a disulfide bond.

7. The composition of claim 1, wherein said removable chemical moiety comprises an azido group.

8. The composition of claim 1, wherein said removable chemical moiety comprises an azidomethyl ether.

9. The composition of claim 5, wherein said label is attached via a cleavable linker.

10. The composition of claim 9, wherein said cleavable linker comprises a disulfide bond.

11. The composition of claim 9, wherein said removable chemical moiety comprises an azido group and said cleavable linker comprises a disulfide bond.

12. The composition of claim 1, further comprising polymerase.