Modified nucleosides and nucleotides and uses thereof

• US 7,816,503 B2
• Filed: 09/21/2009
• Issued: 10/19/2010
• Est. Priority Date: 08/19/2005
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Milton, John, Liu, Xiaohai
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/586,385
• Publication Number:
  US 20100068722A1
• Time in Patent Office: 393 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/26.6, 435/6, 435/91.1
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone

Abstract

The invention is directed to modified guanine-containing nucleosides and nucleotides and uses thereof. More specifically, the invention relates to modified fluorescently labelled guanine-containing nucleosides and nucleotides which exhibit enhanced fluorophore intensity by virtue of reduced quenching effects.

Claims

1. A modified nucleotide or nucleoside comprising a guanine base or a 7-deazaguanine base attached to a fluorophore through a cleavable linking group, wherein said linking group comprises a cleavable functionality and a spacer group of formula —

((CH₂)₂O)_n— and

n is an integer between 2 and 50.

2. The modified nucleotide or nucleoside of claim 1 wherein n is between 5 and 30.

3. The modified nucleotide or nucleoside of claim 1 wherein n is between 10 and 25.

4. The modified nucleotide or nucleoside of claim 1 wherein the cleavable functionality comprises N₃.

5. The modified nucleotide or nucleoside of claim 1 wherein the cleavable linking group is cleavable using a phosphine.

6. The modified nucleotide or nucleoside of claim 1 wherein said nucleotide or nucleoside comprises ribose or 2′

-deoxyribose having a blocking group attached to the 3′

oxygen atom thereof.

7. The modified nucleotide or nucleoside of claim 1 which is a deoxyribonucleotide.

8. A polynucleotide comprising at least one modified nucleotide according to claim 1.

9. A method of detecting a modified guanosine nucleotide incorporated into a polynucleotide which comprises:

(a) incorporating at least one modified nucleotide as defined in claim 1 into a polynucleotide and

(b) detecting the modified nucleotide(s) incorporated into the polynucleotide by detecting the fluorescent signal from said modified nucleotide(s).

10. A method according to claim 9 wherein step (a) comprises incorporating at least one modified nucleotide as into a polynucleotide using a polymerase enzyme.

11. A method according to claim 10 wherein step (a) comprises incubating a template polynucleotide strand with a reaction mixture comprising fluorescently labelled modified nucleotides and a polymerase under conditions which permit formation of a phosphodiester linkage between a free 3′

hydroxyl group on a polynucleotide strand annealed to said template polynucleotide strand and a 5′

phosphate group on said modified nucleotide, wherein said fluorescently labelled modified nucleotides are comprised of a guanine, a 7-deazaguanine base or derivative thereof attached to a fluorophore through a linking group, characterised in that said linking group comprises a spacer group of formula —

((CH₂)₂O)_n—

wherein n is an integer between 2 and 50.

12. A method of sequencing a template nucleic acid molecule comprising:

incorporating one or more nucleotides into a strand of nucleic acid complementary to the template nucleic acid and determining the identity of the base present in one or more incorporated nucleotide(s) in order to determine the sequence of the template nucleic acid molecule;

wherein the identity of the base present in said nucleotide(s) is determined by detecting a fluorescent signal produced by said nucleotide(s);

characterised in that at least one incorporated nucleotide is a modified nucleotide as defined in claim 1.

13. A method according to claim 12 wherein the identity of the base present in said nucleotide(s) is determined after each nucleotide incorporation step.