Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis

Patent metadata

Field	Value
Publication number	US-9670539-B2
Application number	US-201514859853-A
Country	US
Kind code	B2
Filing date	Sep 21, 2015
Priority date	Oct 19, 2007
Publication date	Jun 6, 2017
Grant date	Jun 6, 2017

Abstract

This invention provides novel azido linkers for deoxynucleotide analogs having a detectable marker attached thereto.

First claim

What is claimed is:

1. A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
a) contacting the nucleic acid with (i) at least four different deoxynucleotide triphosphate (dNTP) analogues, each having the structure:

wherein F is a fluorophore, b is a base which is adenine, guanine, cytosine, uracil or thymine, wherein the fluorophore attached through a linker to each type of base differs in its emission or excitation spectra from a fluorophore attached to each of the remaining types of bases, and each of the four dNTP analogues differs from the remaining three dNTP analogues by having a different base, wherein L is an azido cleavable linker molecule or a nitrobenzyl cleavable linker molecule, and R is a cleavable chemical group which is not hydrogen, (ii) a nucleic acid polymerase and (iii) a nucleic acid primer which hybridizes with the nucleic acid,
under conditions permitting one of the four dNTP analogues that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of the nucleic acid primer and thereby extend the primer;

b) identifying the fluorophore of the dNTP analogue which has formed the phosphodiester bond, thereby identifying the consecutive nucleotide;
c) cleaving the linker attaching the fluorophore of the dNTP analogue which has formed the phosphodiester bond and cleaving the cleavable chemical group from the dNTP;
d) iteratively repeating steps a) through c) for each of the consecutive nucleotide residues to be identified until the final consecutive nucleotide residue to be identified in steps a) through e);
e) repeating steps a) and b) to identify the final consecutive nucleotide residue in steps a) through e);
f) denaturing the extended primer so as to de-hybridize it from the nucleic acid;
g) contacting the nucleic acid with (i) three different types of deoxynucleotide triphosphate (dNTP), (ii) a nucleic acid polymerase and (iii) a second nucleic acid primer which hybridizes with the nucleic acid, under conditions permitting one of the three different types of dNTP that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of the second nucleic acid primer and thereby extend the second nucleic acid primer;
h) contacting the nucleic acid with (i) three different types of deoxynucleotide triphosphate (dNTP), wherein at least one of the types of deoxynucleotide triphosphate is not used in step g), under conditions permitting one of the three different types of dNTP that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of the extended second nucleic acid primer and thereby further extend the second nucleic acid primer;
i) repeating steps g) and h) until the second nucleic acid primer is extended up to and including a residue corresponding to the final consecutive nucleotide residue identified in step e);
j) contacting the extended second nucleic acid primer with (i) at least four different deoxynucleotide triphosphate (dNTP) analogues, each having the structure:

wherein F is a fluorophore, b is a base which is adenine, guanine, cytosine, uracil or thymine, wherein the fluorophore attached through a linker to each type of base differs in its emission or excitation spectra from a fluorophore attached to each of the remaining types of bases, and each of the four dNTP analogues differs from the remaining three dNTP analogues by having a different base, wherein L is an azido cleavable linker molecule or a nitrobenzyl cleavable linker molecule, and R is a cleavable chemical group which is not hydrogen, under conditions permitting one of the four dNTP analogues that is complementary to the next consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of the extended second nucleic acid primer and thereby further extend the second primer;

k) identifying the fluorophore of the dNTP analogue which has formed the phosphodiester bond, thereby identifying the consecutive nucleotide;
l) cleaving the fluorophore and the cleavable chemical group from the dNTP analogue which formed the phosphodiester bond so as to thereby permit incorporation of a further dNTP analogue into the extended second nucleic acid primer;
m) iteratively repeating steps j) through l) for each of the consecutive nucleotide residues to be identified until the final consecutive nucleotide residue to be identified in steps j) through n);
n) repeating steps j) and k) to identify the final consecutive nucleotide residue to be identified in steps j) through n);
so as to thereby determining the identity of each of the series of consecutive nucleotide residues in the nucleic acid.

2. The method of claim 1 , wherein in steps g) and h) the three types of dNTPs are chosen from the group dATP, dCTP, dGTP, dTTP or dITP.

3. The method of claim 1 , wherein the linker in each of step a) and j) independently each comprise the structure:

or the structure:

wherein α represents a point of attachment to the base and β represents a point of attachment to the fluorophore, and wherein R is a cleavable chemical group.

4. The method of claim 1 , wherein one or more linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.

5. The method of claim 1 , wherein one or more linkers are photocleavable or chemically cleavable.

6. The method of claim 1 , wherein one or more chemical groups are photocleavable or chemically cleavable.

7. The method of claim 1 , wherein R in the structures set forth in steps a) and/or j) is independently chosen from an —N 3 group or an allyl group.

8. The method of claim 1 , wherein the cleavable chemical group in step g) is independently chosen from an —N 3 group or an allyl group.

9. A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:
a) contacting the nucleic acid with (i) at least four different deoxynucleotide triphosphate (dNTP) analogues, each having the structure:

wherein F is a fluorophore, b is a base which is adenine, guanine, cytosine, uracil or thymine, wherein the fluorophore attached through a linker to each type of base differs in its emission or excitation spectra from a fluorophore attached to each of the remaining types of bases, and each of the four dNTP analogues differs from the remaining three dNTP analogues by having a different base, wherein L is an azido cleavable linker molecule or a nitrobenzyl cleavable linker molecule, and R is a cleavable chemical group which is not hydrogen, (ii) a nucleic acid polymerase and (iii) a nucleic acid primer which hybridizes with the nucleic acid,
under conditions permitting one of the four dNTP analogues that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of the nucleic acid primer and thereby extend the primer;

Assignees

• Univ Columbia

Inventors

• Ju Jingyue
• Cao Huanyan
• Li Zengmin
• Meng Qinglin
• Guo Jia
• Zhang Shenglong
• Yu Lin

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07H19/20

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07H19/16

  Purine radicals · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title