# US 8,148,064 B2 · Labelled nucleotides

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- Number: US 8,148,064 B2
- Document ID: US8148064B2
- Status: Granted
- Kind: B2
- Application number: 12/804,296
- Filed: 2010-07-19
- Published: 2011-07-28
- Granted: 2012-04-03
- Earliest claimed: 2002-08-23
- Assignees: Illumina Cambridge Limited
- Inventors: Shankar Balasubramanian; Colin Barnes; Xiaohai Liu; John Milton
- Web page: https://patentgrep.com/patent/US8148064B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6823: Release of bound markers
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2525/186: incorporating a non-extendable or blocking moiety
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group.

## Claims

### Claim 1

A method for detecting the extension of a primer annealed to a target polynucleotide, comprising:
(a) providing at least one nucleoside comprising
(1) a label attached via a first linker to the nucleoside base, and
(2) a protecting group attached via a second linker to the 2′ or 3′ oxygen of the nucleoside sugar,
wherein the first and second linkers are both cleavable under a single set of conditions;
(b) extending the annealed primer with the nucleoside only if the nucleoside base is complementary to the base at the corresponding position of the target polynucleotide;
(c) detecting the presence of the label on the extended primer; and
(d) incubating the extended primer under the single set of cleavage conditions, whereby both the first and second linkers are cleaved;
thereby detecting the extension of the primer.

### Claim 2 (depends on 1)

The method of claim 1, wherein the target polynucleotide is immobilized.

### Claim 3 (depends on 1)

The method of claim 1, wherein step (a) provides at least two different nucleosides, each having a different label, and the different labels are distinguishable in step (c).

### Claim 4 (depends on 3)

The method of claim 3, wherein step (a) provides four different nucleosides.

### Claim 5 (depends on 1)

The method of claim 1, wherein the nucleoside provided in step (a) is phosphorylated as a nucleotide.

### Claim 6 (depends on 5)

The method of claim 5, wherein the nucleotide provided in step (a) comprises three phosphate groups.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first and/or second linkers comprise an azide.

### Claim 8 (depends on 1)

The method of claim 1, wherein the first and/or second linkers comprise an allylic group.

### Claim 9 (depends on 1)

The method of claim 1, wherein extending step (b) is performed by a DNA polymerase.

### Claim 10 (depends on 1)

The method of claim 1, wherein extending step (b) is performed by a reverse transcriptase.

### Claim 11 (depends on 1)

The method of claim 1, wherein extending step (b) is performed by a terminal transferase.

### Claim 12 (depends on 1)

The method of claim 1, wherein after step (b), any non-incorporated nucleosides are separated from the extended primers.

### Claim 13 (depends on 1)

The method of claim 1, wherein step (d) removes the protecting group to expose a hydroxyl.

### Claim 14 (depends on 1)

The method of claim 1, further comprising:
(e) repeating steps (a), (b), (c), and (d) sequentially.

### Claim 15 (depends on 14)

The method of claim 14, wherein step (e) is repeated more than once.

### Claim 16 (depends on 14)

The method of claim 14, wherein steps (a)-(d) are initially performed with one nucleoside and steps (a)-(d) in step (e) are performed with a different nucleoside.

### Claim 17 (depends on 16)

The method of claim 16, wherein two nucleosides are used initially, and two different nucleosides are used in step (e).