Nucleic-acid probe substrate, system for temperature control of the substrate, and gene detection method making use of the same

• US 7,713,692 B2
• Filed: 06/23/2003
• Issued: 05/11/2010
• Est. Priority Date: 06/24/2002
• Status: Expired due to Fees

• Current Assignee: Canon Ayutthaya Limited (Canon Inc.)
• Original Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Inventors: Kawaguchi, Masahiro
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US10/602,464
• Publication Number:
  US 20040009525A1
• Time in Patent Office: 2,514 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: B01L 2200/026 Fluid interfacing between devices or objects, e.g. connectors, inlet details
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0822 Slides
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2035/00376 Conductive heating, e.g. heated plates

Abstract

A system used to detect and assay genes by using a nucleic-acid probe substrate is disclosed, which can be used in highly general-purpose genetic chips usable for various purposes, and has a temperature control means enabling control of temperature of the whole genetic chip in a high reproducibility and uniformity, and further even satisfies low-cost performance. A method for detecting and assaying gene nucleic acids by using such a system is also disclosed. A heat-conductive material made up using a material having good thermal conductivity, having a shape having general-purpose properties, is used as the temperature control means, and is disposed in such a form that it covers substantially the whole substrate face or stands in contact therewith, on the back of the substrate, or on the side facing the surface to which the detecting single-stranded nucleic acid fragments have been immobilized. This makes it possible to improve the diffusion of heat in the in-plane direction of the whole substrate face. In addition, heat is given and received through such a heat-conductive material.

Claims

1. A system for reaction of a nucleic acid comprising:

a reaction unit comprising;

a nucleic acid probe array substrate having nucleic acid probes immobilized in an array on a surface of the substrate;

a cover member for forming a chamber with said probes immobilized on said surface, wherein a liquid can be filled into the chamber so as to apply said liquid to each of said probes;

a heat conduction member for improving thermal diffusion in the liquid within said chamber, the heat conduction member being in contact with said substrate or said cover member with their contacting surfaces being flat and having no recesses; and

a temperature control block for controlling the temperature of said heat conduction member,

wherein said temperature control block includes a plurality of holes at a contact portion for inserting microtubes thereinto, and

wherein said heat conduction member being for filling the plurality of holes at the contact portion of said temperature control block including a plurality of legs and each of the plurality of legs of said heat conduction member is adapted to be inserted into and in close contact with each one of the plurality of holes at the contact portion of said temperature control block, which is located on a back surface of said substrate, and the temperature control block being in contact with said substrate or said cover member.

2. A system for reaction of a nucleic acid according to claim 1, wherein said temperature control block is a heat block adapted to receive a microtube.

3. The system according to claim 2, wherein said heat conduction member is formed of metal, resin or a composite of metal and resin.

4. A reaction unit for use in the system for reaction according to claim 1.

5. A heat conduction adapter for using a heater with a plurality of holes for microtubes in temperature control for a nucleic acid probe array substrate, the heat conduction adapter comprising:

a face provided with a plurality of legs having the same shape as that of the microtubes, and

another face being flat and having no recesses for contacting with a face of the nucleic acid probe array substrate or a cover forming a chamber with the nucleic acid probe array substrate,

wherein the plurality of legs on the face of the heat conduction adapter are fitted into the plurality of holes of the heat conduction adapter, thus bringing the heater into thermal contact with the nucleic acid probe array.

6. A method for detecting genes by utilizing as a detection means a substrate to the substrate surface of which a plurality of nucleic-acid probes containing single-stranded nucleic acid fragments having a complementary sequence in respect to a target DNA have been immobilized in order that the target DNA contained in a specimen is detected according to hybridization;

the method comprising;

providing the heat conduction adaptor of claim 5;

disposing said heat conduction adaptor on the back of the substrate to the substrate surface of which the plurality of single-stranded nucleic acid fragments have been immobilized, and in contact with the back of the substrate;

disposing a heater or a cooler in contact with said heat conduction adaptor;

providing a temperature controller for controlling the amount of heat flowing across the heater or cooler and said heat conduction adaptor to control the temperature of the heat-conductive material;

providing a specimen comprising the target DNA to the substrate surface comprising the immobilizing nucleic acid fragments;

performing a hybridization reaction; and

detecting the hybridization, the detecting being operated while the substrate standing bonded sandwichedly and the specimen standing in contact with the substrate surface are temperature-controlled through the temperature control of the heat-conductive material by the temperature controller during the operation of gene detection.

7. The method according to claim 6, wherein, in a plurality of steps involved in the gene detection, said substrate and said specimen standing in contact with the substrate surface are temperature-controlled;

and

the temperature in the plurality of steps requiring temperature control is successively controlled by the temperature controller which utilizes said heater.

8. The method according to claim 6, wherein, in a plurality of steps involved in the gene detection, said substrate and said specimen standing in contact with the substrate surface are temperature-controlled;

and

the temperature in the plurality of steps requiring temperature control is successively controlled by the temperature controller which utilizes said cooler.

9. The method according to claim 6, wherein, as said heat-conductive material, which is utilized for the temperature control the substrate and of the specimen standing in contact with the substrate surface, a heat-conductive material is used which is formed of any one of a metal and a resin or a composite of these two or more.

10. A method for detecting genes by utilizing as a detector a substrate to the substrate surface of which a plurality of nucleic-acid probes containing single-stranded nucleic acid fragments having a complementary sequence in respect to a target DNA have been immobilized in order that the target DNA contained in a specimen is detected according to hybridization;

the method comprising;

providing the heat conduction adaptor of claim 5;

disposing said heat conduction adaptor on the surface of the substrate to the substrate surface of which the plurality of single-stranded nucleic acid fragments have been immobilized, facing, and in contact with, the substrate surface, partly leaving a space for feeding the specimen thereinto;

disposing a heater or a cooler in contact with said heat conduction adaptor;

providing a temperature controller for controlling the amount of heat flowing across the heating means or cooling means and said heat conduction adaptor to control the temperature of the heat-conductive material;

providing a specimen comprising the target DNA to the substrate surface comprising the immobilizing nucleic acid fragments;

performing a hybridization reaction; and

detecting the hybridization, the detecting being operated while the specimen fed into the space and the substrate surface, which are in contact with the heat-conductive material, being temperature-controlled through the temperature control of said heat conduction adaptor by the temperature controller during the gene detection.

11. The method according to claim 10, wherein, in a plurality of steps involved in the gene detection, said substrate and said specimen standing in contact with the substrate surface are temperature-controlled;

and

the temperature in the plurality of steps requiring temperature control is successively controlled by the temperature controller which utilizes said heater.

12. The method according to claim 10, wherein, in a plurality of steps involved in the gene detection, said substrate and said specimen standing in contact with the substrate surface are temperature-controlled;

and

the temperature in the plurality of steps requiring temperature control is successively controlled by the temperature controller which utilizes said cooler.

13. The method according to claim 10, wherein, as said heat-conductive material, which is utilized for the temperature of the substrate and the specimen standing in contact with the substrate surface, said heat conduction adaptor is used which is formed of any one of a metal and a resin or a composite of these two or more.