Methods and compositions for incorporating nucleotides

• US 10,472,382 B2
• Filed: 11/16/2018
• Issued: 11/12/2019
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Inventors: Gordon, Steven, Olejnik, Jerzy
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US16/193,639
• Publication Number:
  US 20190119315A1
• Time in Patent Office: 361 Days
• Field of Search: 536 41, 536 261, 536 266
• 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 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.