3'-O-FLUORESCENTLY MODIFIED NUCLEOTIDES AND USES THEREOF

• US 20110076679A1
• Filed: 10/26/2009
• Published: 03/31/2011
• Est. Priority Date: 09/29/2009
• Status: Active Grant

• Current Assignee: Korea Advanced Institute of Science and Technology
• Original Assignee: Korea Advanced Institute of Science and Technology
• Inventors: AHN, He-Chul, AHN, Dae-Ro, SHIN, Dongyun
• Granted Patent: US 8,030,466 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The present invention relates to a DNA sequencing method using a nucleoside triphosphate with a fluorescent blocking group on its 3′-OH end as a reversible terminator. Further, the present invention relates to sequencing-by-synthesis method using the mono-modified reversible terminator (MRT), the novel nucleotide monomer having a reversible fluorescent blocking group removable chemically or enzymatically at its 3′-OH end. The sequencing method of the present invention facilitates sequencing of bases inserted by terminating extension of a nucleotide chain by the nucleotide monomer and then detecting fluorescence signal from 3′-OH end. At this time, after analyzing the fluorescence signal, the blocking group conjugated to the 3′-OH end can be effectively removed, indicating that a free 3′-OH functional group can be successfully restored, so that the next monomer insertion is possible, making continuous sequencing possible.

Claims

1. A nucleotide monomer having a physically or a chemically removable reversible fluorescent group on a 3′

-OH moiety of a nucleoside triphosphate having a ribose or a deoxyribose as a sugar backbone.

2. The nucleotide monomer according to claim 1, wherein the reversible fluorescent group is a fluorophore (FL) itself or composed of the fluorophore and a linker connecting the fluorophore to the 3′

-OH moiety.

3. The nucleotide monomer according to claim 2, wherein the fluorophore is selected from the group consisting of coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, Cy5, Cy3, Texas Red, and their derivatives.

4. The nucleotide monomer according to claim 2, wherein the linker is selected from the group consisting of alkyl, allyl, azidomethyl, disulfide, 2- or 4-nitrobenzyl and a mixture thereof.

5. The nucleotide monomer according to claim 1, wherein the monomer includes the compound represented by [Formula 2]:

6. A sequencing method comprising the following steps:

1) synthesizing the nucleotide monomer of claim 1;

2) extending a primer by one nucleotide using a nucleotide chain of interest as a template, using the nucleotide monomer of step

     1) and a DNA or an RNA polymerase;

3) identifying a base of an extended nucleotide chain by detecting fluorescence signal from the reversible fluorescent blocking group conjugated to a 3′

-OH group of the extended nucleotide chain resulted from polymerization of step

     2);

4) removing the reversible fluorescent blocking group from the extended nucleotide chain of step

     3) and then restoring a free 3′

-OH group to continue polymerization; and

5) repeating steps from

     2) to

     4) to sequence a target template.

7. The sequencing method according to claim 6, wherein the template nucleic acid chain of step 2) is selected from the group consisting of genomic DNAs, plasmids and oligonucleotides.

8. The sequencing method according to claim 6, wherein the polymerase of step 2) is selected from the group consisting of a thermophilic polymerase, a mesophilic polymerase and its mutant.

9. The sequencing method according to claim 6, wherein the fluorescence signal of step 3) is detected by measuring fluorescence of a solid phase on which the double stranded DNA of template and the primer is immobilized or by measuring fluorescence of solution containing the double stranded DNA.

10. The sequencing method according to claim 6, wherein the reversible fluorescent group of step 4) is preferably removed by using chemicals or enzymes or irradiation.

11. A sequencing kit for sequencing-by-synthesis (SBS) containing the nucleotide monomer of claim 1 or its multimer.