DNA sequencing by nanopore using modified nucleotides

• US 8,889,348 B2
• Filed: 06/07/2007
• Issued: 11/18/2014
• Est. Priority Date: 06/07/2006
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue
• Primary Examiner(s): KAPUSHOC, STEPHEN THOMAS
• Application Number:
  US12/308,091
• Publication Number:
  US 20090298072A1
• Time in Patent Office: 2,721 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

This invention provides a process for sequencing single-stranded DNA by employing a nanopore and modified nucleotides.

Claims

1. A method for determining the nucleotide sequence of a single-stranded DNA comprising the steps of:

(a) synthesizing a precursor of the single-stranded DNA as part of a double-stranded DNA using a single-stranded DNA template, a primer, a DNA polymerase, and all four nucleotides present in DNA, wherein each A or each G residue, but not both, comprise a first label bound to its respective base, and each C or each T residue, but not both, comprise a second label bound to its respective base;

(b) denaturing the double-stranded DNA obtained in (a);

(c) isolating the single-stranded DNA to be sequenced comprising the nucleotides containing the first label and the second label from the denatured double-stranded DNA;

(d) reacting the single-stranded DNA isolated in (c) with first modifying groups which form bonds with the first labels and second modifying groups which form bonds with the second labels, so as to obtain the single-stranded DNA to be sequenced wherein each labeled nucleotide is conjugated to a modifying group;

(e) passing the single-stranded DNA to be sequenced through a pore of suitable diameter by applying and electric field to the DNA;

(f) determining an electronic signature for each nucleotide of the single-stranded DNA which passes through the pore; and

(g) comparing each electronic signature determined in (f) with electronic signatures corresponding to each of A, G, C, and T or corresponding to the bases conjugated to the modifying group, so as to determine the identity of each such nucleotide,

thereby determining the nucleotide sequence of the single-stranded DNA.

2. The method of claim 1, wherein the pore has a diameter of from about 1 nm to about 5 nm.

3. The method of claim 1, wherein the pore has a diameter of from about 1 nm to about 3 nm.

4. The method of claim 1, wherein the pore has a diameter of about 1 nm, 2 nm, 3 nm, 4 nm or 5 nm.

5. The method of claim 1, wherein each A and each T nucleotide comprises a modifying group bound to its labeled base.

6. The method of claim 1, wherein each G and each C nucleotide comprises a modifying group bound to its labeled base.

7. The method of claim 1, wherein each A and each C residue or each G and each T residue comprises a modifying group bound to its respective base.

8. The method of claim 1, wherein 5′

-end of the primer comprises a biotin moiety.

9. The method of claim 1, wherein the first label is an amino group and the second label is an azido group.

10. The method of claim 1, wherein the first label is an azido group and the second label is an amino group.

11. A method for determining the nucleotide sequence of a single-stranded DNA comprising the steps of:

(a) passing the single-stranded DNA through a nanopore of a suitable diameter by applying an electric field, wherein each A or each G residue, but not both, comprises a modifying group bound to a label added to its respective base, and each C or each T residue, but not both, comprises a modifying group bound to a label added to its respective base, and each type of nucleotide in the single-stranded DNA has a characteristic electronic signature which is distinguishable from the electronic signature of all other types of nucleotides in the single-stranded DNA;

(b) determining the electronic signature for each nucleotide within the single-stranded DNA which passes through the nanopore

(c) comparing each electronic signature determined in step (b) with the electronic signature characteristic of each type of nucleotide so as to determine the identity of each such nucleotide that passes through the nanopore;

thereby determining the nucleotide sequence of the single-stranded DNA.

12. The method of claim 11, wherein the nanopore has a diameter of from about 1 nm to about 5 nm.

13. The method of claim 11, wherein the single-stranded DNA whose sequence is to be determined is obtained by:

(a) synthesizing the single-stranded DNA as part of a double-stranded DNA using a single-stranded DNA template, a primer, a DNA polymerase, and nucleotides, wherein each A or each G residue, but not both, comprises a modifying group bound to a label added to its respective, and each C or each T residue, but not both, comprises a modifying group bound to a label added to its respective base, wherein all of the A or G nucleotides, and all of the C or the T nucleotides within the single-stranded DNA comprise an identical modifying group bound to the label added to their respective bases and each type of nucleotide has a characteristic electronic signature which is distinguishable from the electronic signature of each other type of nucleotide in the DNA;

(b) denaturing the double-stranded DNA obtained in step (a); and

(c) isolating the single-stranded DNA to be sequenced comprising modified nucleotides from the denatured double-stranded DNA.

14. The method of claim 11, wherein the single-stranded DNA whose sequence is to be determined is obtained by:

(a) synthesizing a precursor of the single-stranded DNA as part of a double-stranded DNA using a single-stranded DNA template, a primer, a DNA polymerase, and all four nucleotides present in DNA, wherein each A or each G residue, but not both, comprise a first label bound to its respective base, and each C or each T residue, but not both, comprise a second label bound to its respective base;

(b) denaturing the double-stranded DNA obtained in (a);

(c) isolating the single-stranded DNA to be sequenced comprising the first label and the second label-containing nucleotides from the denatured double-stranded DNA; and

(d) reacting the single-stranded DNA isolated in (c) with first modifying groups which form bonds with the first labels and second modifying groups which form bonds with the second labels,

so as to obtain the single-stranded DNA to be sequenced.

15. The method of claim 14, wherein the first label is an amino group and the second label is an azido group.

16. The method of claim 14, wherein the first label is an azido group and the second label is an amino group.

17. The method of claim 11, wherein each A nucleotide of said single stranded DNA comprises a modifying group bound to its labeled base.

18. The method of claim 11, wherein each T nucleotide of said single stranded DNA comprises a modifying group bound to its labeled base.

19. The method of claim 11, wherein A, G, C, and T nucleotides of said single-stranded DNA have an order of bulkiness that is T>

A>

G>

C.