DNA sequencing with reagent recycling on wiregrid

Patent metadata

Field	Value
Publication number	US-9823196-B2
Application number	US-201314371890-A
Country	US
Kind code	B2
Filing date	Jan 9, 2013
Priority date	Jan 13, 2012
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

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 moietys. 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,
an optical arrangement,
wherein the optical arrangement is configured to emit cleavage light of a cleavage wavelength λ CL , wherein the cleavage light is polarized light,
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 the cleavage light of the 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 confine the excitation light,
wherein the substrate is configured to provide for an evanescent wave of the excitation light at the first surface of the substrate,
wherein the substrate is configured to confine the cleavage light,
wherein the substrate is configured to provide for an evanescent wave of the cleavage light at the first surface of the substrate,
wherein the evanescent wave of the cleavage light induces the cleavage reaction, and
wherein the substrate comprises a wiregrid including wires that are opaque.

2. The device according to claim 1 , the device further comprising
a solution with a plurality of nucleotides and an enzyme,
wherein the nucleotides respectively comprise a blocking moiety, wherein the 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 chosen from the group comprising 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 cleaving reaction takes a time t cleavage ,
wherein the cleaving 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 along the first direction, 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 wiregrid forms slit-apertures, wherein each of the slit-apertures has a size that is less than λ CL /2NA, and wherein NA is the numerical aperture of the respective aperture.

9. The device according to claim 1 , wherein the wiregrid forms a plurality of apertures and wherein the at least one molecule is disposed within at least one of said apertures during the cleavage reaction.

10. The device of claim 9 wherein the plurality of apertures are slit-apertures, wherein each of the slit-apertures have a size that is less than λ CL /2NA, and wherein NA is the numerical aperture of the respective slit-aperture.

11. The device of claim 1 , wherein the wiregrid is configured reflect the cleavage light and to transmit light having a polarization that is different from a polarization of the cleavage light.

12. A method for optically controlling an iterative stepwise reaction to determine a sequence of a nucleic acid by synthesis, the method comprising the steps:
providing a substrate with a molecule bound on a first surface of the substrate,
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,
confining the excitation light by the substrate thereby providing for an evanescent wave of the cleavage 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 nucleotide, wherein the cleavage light is polarized light, and
confining the cleavage light of the cleavage wavelength λ CL by the substrate thereby providing for an evanescent wave of the cleavage light by the substrate at the first surface of the substrate, wherein the evanescent wave of the cleavage light induces the cleavage reaction and wherein the substrate comprises a wiregrid including wires that are opaque.

13. The method according to claim 12 , the method further comprising the steps:
providing for a solution with a plurality of nucleotides and an enzyme, wherein the nucleotides respectively comprise a blocking moiety which comprises the fluorescent label,
blocking a synthesizing activity of the enzyme by the blocking moiety when the respective nucleotide is incorporated into the molecule bound to the first surface, and wherein the step of inducing the cleaving reaction is performed such that the blocking moiety comprising the fluorescent label is cleaved away from the incorporated nucleotide.

14. The method according to claim 12 , wherein the blocking moiety is chosen from the group comprising 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.

15. The method according to claim 12 , 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

Assignees

• Koninklijke Philips Nv

Inventors

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

Classifications

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• B01J2219/00711

  Light-directed synthesis · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2523/319

  Photocleavage, photolysis, photoactivation · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title