3′-o-fluorescently modified nucleotides and uses thereof
View Patent ↗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.
1. A nucleotide monomer having a physically or a chemically removable reversible fluorescent blocking group on a 3′-OH moiety of a nucleoside triphosphate in a compound represented by Formula 2:
wherein:
R 1 is triphosphate;
R 2 is nucleobase;
R 4 is H or OH; and
the linker-R 3 is selected from the group consisting of the fluorescent blocking groups having the following chemical structures:
wherein R 5 is fluorophore.
2. The nucleotide monomer according to claim 1 , wherein the compound has the following chemical structure:
wherein
R 1 is triphosphate;
R 2 is nucleobase;
R 4 is H or OH; and
R 5 is fluorophore.
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 1 , wherein the compound has the following chemical structure:
wherein
R 1 is triphosphate;
R 2 is nucleobase; and
R 4 is H or OH.
5. A sequencing method comprising the following steps:
1) synthesizing the nucleotide monomer of claim 1 ;
2) preparing a nucleotide chain of interest as template and a primer
3) incorporating one nucleotide monomer of step 1) into the primer of step 2) by a DNA or an RNA polymerase, resulting in an extended primer;
4) identifying a base of the extended primer by detecting fluorescence signal from the fluorescent blocking group conjugated to a 3′-OH moiety of the extended primer resulted from the incorporating of step 3);
5) removing the fluorescent blocking group from the extended primer of step 4) and then restoring a free 3′-OH group to continue incorporation of the nucleotide monomer of step 1); and
6) repeating steps from 3) to 5) to sequence a target template.
6. The sequencing method according to claim 5 , wherein the template of step 2) is selected from the group consisting of genomic DNAs, plasmids and oligonucleotides.
7. The sequencing method according to claim 5 , wherein the polymerase of step 3) is selected from the group consisting of a thermophilic polymerase, a mesophilic polymerase and its mutant.
8. The sequencing method according to claim 5 , wherein the fluorescence signal of step 4) 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.
9. The sequencing method according to claim 5 , wherein the fluorescent blocking group of step 5) is preferably removed by using Pd catalyst, tris-alkylphosphine, tris-arylphosphine or their derivatives.
10. A sequencing kit for sequencing-by-synthesis (SBS) containing the nucleotide monomer of claim 1 or its multimer.
11. The nucleotide monomer according to claim 1 , wherein the fluorophore is selected from the group consisting of coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, Cy5, Cy3, Texas Red, and their derivatives.
12. The nucleotide monomer according to claim 1 , wherein the compound has the following chemical structure:
wherein
R 1 is triphosphate;
R 2 is nucleobase;
R 4 is H or OH; and
R 5 is fluorophore.
13. The nucleotide monomer according to claim 12 , wherein the fluorophore is selected from the group consisting of coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, Cy5, Cy3, Texas Red, and their derivatives.