Nucleic acid analysis device

Patent metadata

Field	Value
Publication number	US-9770713-B2
Application number	US-201314421239-A
Country	US
Kind code	B2
Filing date	Jul 8, 2013
Priority date	Aug 30, 2012
Publication date	Sep 26, 2017
Grant date	Sep 26, 2017

Abstract

A nucleic acid analysis device which can determine a DNA sequence has a flowcell in which two or more DNA fragment clusters of two or more DNA fragments having identical nucleotide sequences are immobilized. At least a part of the flowcell is made of a transparent material. An irradiation unit irradiates a part in which the DNA fragment clusters are immobilized. The device has a lens for collecting fluorescence, and a light-detection element. A solution containing only dATP having a fluorescently modified phosphate terminal among four bases, a solution containing only dCTP having a fluorescently modified phosphate terminal among the four bases, a solution containing only dGTP having a fluorescently modified phosphate terminal among the four bases, a solution containing only dTTP having a fluorescently modified phosphate terminal among the four bases, and a buffer solution are sent sequentially to where the DNA fragment clusters are immobilized.

First claim

The invention claimed is:

1. A DNA sequencing device for sequencing DNA without a terminator and in real-time, comprising:
a flowcell in which two or more DNA fragment clusters of two or more DNA fragments having identical nucleotide sequences are immobilized, wherein at least a part of the flowcell where the two or more DNA fragment clusters are immobilized is made of a transparent material;
an irradiation light source to irradiate the part of the flowcell in which the DNA fragment clusters are immobilized;
a first lens to collect fluorescence from the part of the flowcell in which the DNA fragment clusters are immobilized; and
a light-detection sensor to detect collected light from the lens; a plurality of solution tanks connected to the flow cell, the solution tanks including a first tank storing a dATP solution containing dATP having a fluorescently modified phosphate terminal, a second tank storing a dCTP solution containing dCTP having a fluorescently modified phosphate terminal, a third tank storing a dGTP solution containing dGTP having a fluorescently modified phosphate terminal, a fourth tank storing a dTTP solution containing dTTP having a fluorescently modified phosphate terminal, and a fifth tank storing a buffer solution for washing out the flowcell;
one or more pumps and valves connected to the flow cell and the solution tanks to send the dATP solution, the dCTP solution, the dGTP solution, the dTTP solution, and the buffer solution to the flowcell; and
a controller which is programmed to fill the part of the flowcell where the two or more DNA fragment clusters are immobilized with the buffer solution, and sequentially send the dATP solution, the dCTP solution, the dGTP solution, and the dTTP solution to the part of the flowcell where the DNA fragment clusters are immobilized,
wherein the controller is further programmed to sequence the DNA fragment clusters in parallel by processing the collected light from the lens when each of the dATP solution, the dCTP solution, the dGTP solution, and the dTTP solution are sequentially sent to the part of the flowcell where the DNA fragment clusters are immobilized, and
wherein the dATP solution, the dCTP solution, the dGTP solution, and the dTTP solution are sequentially sent a plurality of times, and
the DNA sequencing device further comprising:
a driving unit on which the flowcell is fixed, and
wherein the controller is further programmed to control the driving unit to drive the flowcell so that a head of each of the dATP solution, the dCTP solution, the dGTP solution, and the dTTP solution in the flowcell substantially corresponds to a light-detection field of the light-detection sensor.

2. The DNA sequencing device of claim 1 ,
wherein the solution tanks are disposed on the flowcell.

3. The DNA sequencing device of claim 1 ,
wherein the controller is further programmed to control the light-detection sensor to detect a change in fluorescence of the part of the flowcell where the DNA fragment clusters are immobilized before and after sending each of the dATP solution, the dCTP solution, the dGTP solution, and the dTTP solution.

4. The DNA sequencing device of claim 1 ,
wherein the flowcell has two or more flow paths.

5. The DNA sequencing device of claim 1 , wherein
the flowcell has two or more flow paths.

6. The DNA sequencing device of claim 2 ,
which has a power supply for applying a voltage between the containers, and
which is characterized in that the solutions are sent by electrophoresis between the containers.

7. The DNA sequencing device of claim 6 ,
which has two or more flow paths and a driving unit for driving the flowcell,
wherein the flowcell is driven in a direction opposite to a solution-sending direction at a speed same as a solution-sending speed and then a light-detection field is moved to another flow path.

8. The DNA sequencing device of claim 1 , further comprising:
a second lens disposed between the first lens and the light-detection sensor.

9. The DNA sequencing device of claim 1 ,
which is characterized in that the dATP solution contains non-modified dATP, the dCTP solution contains non-modified dCTP, the dGTP solution contains non-modified dGTP, and the dTTP solution contains non-modified dTTP.

10. The DNA sequencing device of claim 1 ,
wherein the solution tanks further include a sixth solution tank containing a non-modified dATP solution, a seventh solution tank containing a non-modified dCTP solution, an eighth solution tank containing a non-modified dGTP solution, and a ninth solution tank containing a non-modified dTTP solution,
wherein the controller is further programmed to send the non-modified dATP solution, the non-modified dCTP solution, the non-modified dGTP solution, and the non-modified dTTP solution to the flowcell.

11. The DNA sequencing device of claim 1 ,
which is characterized in that the flowcell has two or more flow paths,
the nucleic acid analysis device has a driving unit for driving the flowcell,
the flowce comprises sample substrate and
the nucleic acid analysis device repeats a process 1 for obtaining an image of fluorescence from the sample substrate surface surrounded by one of the flow paths, a process 2 for moving a light-detection field to the substrate surface surrounded by another flow path by the driving unit after the process 1, and a process 3 for obtaining an image of fluorescence from the substrate surface surrounded by the flow path.

12. The DNA sequencing device of claim 1 ,
which further has an optical element which reflects a light emitted from the irradiation unit and transmits the fluorescence,
the flowcell comprises sample substrate and
which is characterized in that the light emitted from the irradiation unit is reflected by the optical element, then passes through the lens and irradiates the sample substrate surface.

13. The DNA sequencing device of claim 1 ,
which is characterized in that the flowcell has a substrate and a light-blocking film,
the light-blocking film has an opening of 500 nm or less,
the substrate at the bottom of the opening is transparent, and
the clusters are immobilized on the bottom of the opening.

14. The DNA sequencing device of claim 1 ,
which further has two or more luminophores disposed in the clusters and
which is characterized in that fluorophores attached to the bases absorb energy from the luminophores and emit fluorescence.

15. The DNA sequencing device of claim 1 ,
wherein a light from the irradiation light source enters the flowcell from a surface opposite to a side of the flowcell at which the DNA fragment clusters are immobilized and a near field is generated at the side at which the DNA fragment clusters are immobilized.

16. The DNA sequencing device of claim 1 ,
wherein the controller is further programmed to calculate an average derivative of a fluorescence intensity from the collected light when each of the dATP solution, the dCTP solution, the dGTP solution, and the dTTP solution are sequentially sent to the part of the flowcell where the DNA fragment clusters are immobilized.

Assignees

• Hitachi High Tech Corp

Inventors

• Haga Takanobu
• Kodama Yoshitaka
• Shoji Tomohiro
• Muramatsu Takamichi
• Yamamoto Shuhei

Classifications

• B01L2300/0636

  Integrated biosensor, microarrays · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• G01N21/6428 Primary

  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

• C12Q2535/122

  Massive parallel sequencing · CPC title

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title