Modified nucleosides and nucleotides and uses thereof

• US 7,592,435 B2
• Filed: 07/27/2006
• Issued: 09/22/2009
• Est. Priority Date: 08/19/2005
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Milton, John, Liu, Xiaohai
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/494,279
• Publication Number:
  US 20070042407A1
• Time in Patent Office: 1,153 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 consisting of a guanine base or a 7-deazaguanine base attached to a fluorophore through a linking group, one or more phosphate groups, and a ribose or 2′

-deoxyribose having a blocking group attached to the 3′

oxygen atom thereof, wherein said linking group comprises a spacer group of formula —

((CH₂)₂O)_n—

wherein n is an integer between 10 and 30, and wherein the presence of the linking group reduces fluorescent signal quenching of the fluorophore by the guanine base or 7-deazaguanine base.

2. The modified nucleotide of claim 1 wherein n is between 10 and 25.

3. The modified nucleotide of claim 1 wherein said linking group is cleavable.

4. The modified nucleotide of claim 3 wherein said linking group and said blocking group are cleavable.

5. The modified nucleotide of claim 1 which is a deoxyribonucleotide.

6. A method of detecting a nucleotide incorporated into a polynucleotide which comprises:

(a) incorporating at least one modified nucleotide as defined in claim 1 into a polynucleotide, wherein said incorporating refers to joining the modified nucleotide to the free 3′

hydroxyl group of a nucleic acid strand via formation of a phosphodiester linkage and

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

7. A method according to claim 6 wherein step (a) comprises incorporating at least one modified nucleotide into a polynucleotide using a polymerase enzyme.

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

incorporating one or more modified nucleotides of claim 1 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).

9. A method according to claim 8 wherein the identity of the base present in said nucleotide(s) is determined after each nucleotide incorporation step.

10. A modified nucleotide consisting of a guanine base or a 7-deazaguanine base attached to a fluorophore through a linking group, one or more phosphate groups, and a ribose, 2′

-deoxyribose or 2′

, 3′

dideoxyribose, wherein said linking group comprises a spacer group of formula —

((CH₂)₂O)_n—

wherein n is an integer between 10 and 30, and wherein the presence of the linking group reduces fluorescent signal quenching of the fluorophore by the guanine base or 7-deazaguanine base.

11. A method of detecting a nucleotide incorporated into a polynucleotide which comprises:

(a) incorporating at least one modified nucleotide as defined in claim 10 into a polynucleotide, wherein said incorporating refers to joining the modified nucleotide to the free 3′

hydroxyl group of a nucleic acid strand via formation of a phosphodiester linkage and

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

12. The method according to claim 11 wherein step (a) comprises incorporating at least one modified nucleotide into a polynucleotide using a polymerase enzyme.

13. The method according to claim 11 wherein n is between 10 and 25.

14. The method according to claim 11 wherein said linking group is cleavable.