Methods and solutions for inhibiting undesired cleaving of labels

• US 9,926,595 B2
• Filed: 03/22/2017
• Issued: 03/27/2018
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: Intelligent Bio-Systems Incorporated. (Qiagen NV)
• Inventors: Olejnik, Jerzy, Guggenheim, Evan, Meyyappan, Visalakshi
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/466,237
• Publication Number:
  US 20170275671A1
• Time in Patent Office: 370 Days
• Field of Search: 435 61
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

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 of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, a cleaving agent scavenger, and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label attached through a cleavable disulfide linker and contains a removable chemical moiety capping the 3′

-OH group, wherein said removable chemical moiety comprises a disulfide bond;

b) incorporating a first nucleotide analogue with said polymerase;

c) detecting the label of the incorporated nucleotide analogue;

d) removing the unique label and the chemical moiety of the incorporated nucleotide analogue capping the 3′

-OH group with said cleaving agent; and

e) introducing said cleaving agent scavenger under conditions such that it reacts with any leftover cleaving agent.

2. The method of claim 1, wherein said cleaving agent is a phosphine.

3. The method of claim 2, wherein said phosphine is Tris(2-carboxy-ethyl)phosphine.

4. The method of claim 1, wherein said cleaving agent scavenger does not contain a nucleic acid base.

5. The method of claim 1, further comprising, prior to step b) hybridizing a primer to said plurality of nucleic acid template molecules, such that said first nucleotide analogue is incorporated into said primer at step b).

6. A method of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, a cleaving agent scavenger, and a plurality of nucleotide analogues selected from the group consisting of cytosine, thymine, deaza-adenine and deaza-guanine, wherein each nucleotide analogue comprises a unique label attached through a cleavable disulfide linker to a 5-position of cytosine or thymine or to a 7-position of deaza-adenine or deaza-guanine, and wherein each nucleotide analogue contains a removable chemical moiety capping the 3′

-OH group, wherein said removable chemical moiety comprises a disulfide bond;

b) incorporating a first nucleotide analogue with said polymerase;

c) detecting the label of the incorporated nucleotide analogue;

d) removing the chemical moiety of the incorporated nucleotide analogue capping the 3′

-OH group and cleaving the cleavable linker with said cleaving agent; and

e) introducing said cleaving agent scavenger under conditions such that it reacts with any leftover cleaving agent.

7. The method of claim 6, further comprising, prior to step b) hybridizing a primer to said plurality of nucleic acid template molecules, such that said first nucleotide analogue is incorporated into said primer at step b).

8. The method of claim 6, wherein said cleaving agent is a phosphine.

9. The method of claim 8, wherein said phosphine is Tris(2-carboxy-ethyl)phosphine.

10. The method of claim 6, wherein said cleaving agent scavenger does not contain a nucleic acid base.