DNA sequencing with reagent recycling on wiregrid

Patent metadata

Field	Value
Publication number	US-10564104-B2
Application number	US-201715806473-A
Country	US
Kind code	B2
Filing date	Nov 8, 2017
Priority date	Jan 13, 2012
Publication date	Feb 18, 2020
Grant date	Feb 18, 2020

Abstract

The present invention relates to DNA sequencing with reagent cycling on the wiregrid. The sequencing approach suggested with which allows to use a single fluid with no washing steps. Based on strong optical confinement and of excitation light and of cleavage light, the sequencing reaction can be read-out without washing the surface. Stepwise sequencing is achieved by using nucleotides with optically cleavable blocking moieties. After read-out the built in nucleotide is deblocked by cleavage light through the same substrate. This ensures that only bound nucleotides will be unblocked.

First claim

The invention claimed is:

1. A device for optically controlling an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis, the device comprising:
a substrate for binding at least one molecule on a first surface of the substrate; and
an optical arrangement comprising a filter disposed on said first surface,
wherein the optical arrangement is configured to direct excitation light of at least a first excitation wavelength λ Ex1 to the substrate to excite a fluorescent label of a first nucleotide,
wherein the first nucleotide is incorporated into the molecule bound on the first surface of the substrate,
wherein the optical arrangement is configured to receive and detect fluorescent light emitted by the fluorescent label of the first nucleotide,
wherein the optical arrangement is configured to direct cleavage light of a cleavage wavelength λ CL to the substrate to optically induce a cleavage reaction at the first nucleotide to cleave a blocking moiety and the fluorescent label away from the first nucleotide,
wherein the substrate is configured to provide for an evanescent wave of the excitation light at the first surface of the substrate,
wherein at least one of the excitation light or the cleavage light is polarized light,
wherein said filter is configured to reflect said at least one of the excitation light or the cleavage light and to transmit light having a polarization that is different from a polarization of said at least one of the excitation light or the cleavage light.

2. The device according to claim 1 , the device further comprising:
a solution with a plurality of nucleotides and an enzyme,

wherein the nucleotides of the solution respectively comprise a respective blocking moiety,
wherein the respective blocking moiety is configured to block a synthesizing activity of the enzyme when the respective nucleotide is incorporated into the molecule bound to the first surface.

3. The device according to claim 2 , wherein the blocking moiety is a photo cleavable 3′-unblocked reversible terminator.

4. The device according to claim 2 , wherein the blocking moiety is selected from the group consisting of a derivative of nitrophenylethyl, 5-methyl(2-(2-nitrophenyl)propyl) carbonate-dUTP analogue, 5-methyl(2-oxo-1,2-diphenylethyl) carbonate-dUTP analog, and any combination thereof.

5. The device according to claim 1 ,
wherein the cleavage reaction takes a time t cleavage ,
wherein the cleavage reaction time t cleavage depends on an intensity of the irradiated cleavage light,
wherein incorporating a second nucleotide into the bound molecule takes a time t incorporation , and
wherein the optical arrangement is configured to provide the irradiated cleavage light with an intensity such that t cleavage <t incorporation .

6. The device according to claim 1 ,
wherein the substrate comprises several adjacent binding positions for binding molecules to the first surface along a first direction,
wherein the device is configured to perform an optical scan by implementing a relative movement between the substrate and the optical arrangement, and
wherein the device is configured to perform the optical scan such that each binding position is firstly irradiated with the excitation light of at least the first wavelength λ Ex1 and subsequently and secondly irradiated with the cleavage light of the cleavage wavelength λ CL in a movement along the first direction.

7. The device according to claim 1 ,
wherein the device is configured to stepwisely and optically induce the incorporation into the bound molecule of nucleotides with a sequence, which is complementary to a sequence of nucleotides of the bound molecule,
wherein the device is configured to stepwisely and optically read out and determine the sequence of nucleotides which are incorporated into the bound molecule, and
wherein the device is configured to base the determination of the sequence of the incorporated nucleotides on the received and detected respective fluorescent light emitted by the fluorescent label of the respective incorporated nucleotide.

8. The device according to claim 1 , wherein the substrate is configured to confine the excitation light, wherein the substrate is configured to confine the cleavage light, and wherein the substrate is configured to provide for an evanescent wave of cleavage light at the first surface of the substrate.

9. A method for optically controlling an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis, the method comprising:
providing a substrate with a molecule bound on a first surface of the substrate, wherein a filter is disposed on said first surface;
irradiating the substrate with excitation light of at least a first excitation wavelength λ Ex1 by an optical arrangement and thereby optically exciting a fluorescent label of a first nucleotide, wherein the first nucleotide is incorporated in the bound molecule on the substrate;
providing an evanescent wave of the excitation light by the substrate at the first surface of the substrate;
receiving and detecting fluorescence of the excited fluorescent label of the first nucleotide by the optical arrangement;
irradiating the substrate with cleavage light of a cleavage wavelength λ CL by the optical arrangement and thereby optically inducing a cleaving reaction at the first incorporated nucleotide, wherein at least one of the excitation light or the cleavage light is polarized light; and
reflecting said at least one of the excitation light or the cleavage light by said filter, wherein the filter is configured to transmit light having a polarization that is different from a polarization of said at least one of the excitation light or the cleavage light.

10. The method according to claim 9 , the method further comprising:
providing a solution with a plurality of nucleotides and an enzyme, wherein the nucleotides of the solution respectively comprise a respective blocking moiety; and
blocking a synthesizing activity of the enzyme by the respective blocking moiety when the respective nucleotide is incorporated into the molecule bound to the first surface, wherein the cleaving reaction is performed such that the respective blocking moiety is cleaved away from the respective nucleotide.

11. The method according to claim 9 , wherein the blocking moiety is selected from the group consisting of a derivative of nitrophenylethyl, 5-methyl(2-(2-nitrophenyl)propyl) carbonate-dUTP analogue, 5-methyl(2-oxo-1,2-diphenylethyl) carbonate-dUTP analog, and any combination thereof.

12. The method according to claim 9 , wherein the substrate comprises several adjacent molecule binding positions at which a molecule is respectively bound to the first surface along a first direction, the method further comprising:
performing an optical scan by implementing a relative movement between the substrate and the optical arrangement, wherein the optical scan is performed such that each bound molecule is firstly irradiated with the excitation light of at least the first excitation wavelength λ Ex1 and subsequently and secondly irradiated with the cleavage light of the cleavage wavelength λ CL in a movement along the first direction.

13. The method according to claim 12 ,
wherein the cleaving reaction takes a time t cleavage ,
wherein the cleaving reaction time t cleavage depends on an intensity of the irradiated cleavage light, the method further comprising:
incorporating a second nucleotide into the bound molecule, wherein the incorporation takes a time t incorporation ; and
selecting the intensity of the irradiated cleavage light at the optical arrang

Assignees

• Koninklijke Philips Nv

Inventors

• Wimberger-Friedl Reinhold
• Lub Johan
• Van Der Zaag Pieter Jan

Classifications

• B01J2219/00605

  the compounds being directly bound or immobilised to solid supports · CPC title

• C12Q2565/601

  being a microscope, e.g. atomic force microscopy [AFM] · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• B01J2219/00722

  Nucleotides · CPC title

• G01N2201/06113

  Coherent sources; lasers · CPC title