Methods And Compositions For Incorporating Nucleotides

• US 20180251832A1
• Filed: 03/05/2018
• Published: 09/06/2018
• 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
• Granted Patent: US 10,329,611 B2
• Time in Patent Office: Days
• 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 method for determining an identity of a nucleotide at a position, comprising:

a) providing a device capable of measuring color in more than one detector channel, 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 capable of being detected in two channels;

b) incorporating, with said polymerase, a first nucleotide analogue from said mixture into said nucleic acid at a position;

c) detecting the label of said incorporated first nucleotide analogue in two channels with said device; and

d) determining the identity of the incorporated nucleotide at said position.

2. 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.

3. The method of claim 1, wherein said labeled reversible terminators are labeled with at least one type of fluorescent dye and said detecting of step c) comprising detecting color of said at least one type of fluorescent dye in two channels.

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

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

6. The method of claim 1, wherein said nucleic acid comprises primer hybridized to template.