Hydroxymethyl Linkers For Labeling Nucleotides

• US 20140045175A1
• Filed: 07/25/2013
• Published: 02/13/2014
• Est. Priority Date: 04/23/2009
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: Intelligent Bio-Systems Incorporated. (Qiagen NV)
• Inventors: Olejnik, Jerzy
• Granted Patent: US 9,322,050 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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 1/6869 Methods for sequencing
• 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 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

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, and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′

-OH group, wherein the label is attached to the base through a chemically cleavable linker, said linker created via reaction with a hydroxymethyl group;

b) incorporating a first nucleotide analogue with said polymerase;

c) detecting the label of the incorporated nucleotide analogue, and

d) chemically cleaving said linker such that said label is removed from said base.

2. The method of claim 1, wherein said label is selected from the group consisting of 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene, Rhodamine, Carboxyrhodamine and Cyanine.

3. The method of claim 1, wherein said removable chemical moiety capping the 3′

-OH group comprises an azido group.

4. The method of claim 1, wherein said removable chemical moiety capping the 3′

-OH group comprises a disulfide bond.

5. The method of claim 1, wherein after said chemical cleaving of step d) said linker consists of a hydroxymethyl group attached to said base.

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

a) providing a plurality of nucleic acid template molecules, a polymerase, and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′

-OH group, wherein the label is attached to the base through a chemically cleavable linker;

b) incorporating a first nucleotide analogue with said polymerase;

c) detecting the label of the incorporated nucleotide analogue, and

d) chemically cleaving said linker such that said label is removed from said base, wherein after said cleaving said linker consists of a hydroxymethyl group attached to said base.

7. The method of claim 6, wherein after said cleaving of step d), said nucleotide has the general structure selected from the group consisting of: