Pcr method by electrostatic transportation, hybridization method for electrostatic transportation and devices therefor

• US 20050064423A1
• Filed: 01/08/2003
• Published: 03/24/2005
• Est. Priority Date: 01/08/2002
• Status: Abandoned Application

• Current Assignee: Japan Science & Technology Trading Co. Limited
• Original Assignee: Japan Science & Technology Trading Co. Limited
• Inventors: Higuchi, Toshiro, Taniguchi, Tomohiro, Torii, Toru, Katayama, Tetsuo
• Application Number:
  US10/499,827
• Publication Number:
  US 20050064423A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 2035/00366 Several different temperatures used
• CPC Class Codes: G01N 2035/00376 Conductive heating, e.g. heated plates

Abstract

To provide a PCR method utilizing electrostatic transportation which allows separate control of individual DNA-containing droplets by accurately transporting the droplets, suitably controlling the temperature thereof and allowing a primer to react therewith; a hybridization method utilizing electrostatic transportation which allows rapid and easy detection of hybridization; and devices therefor.

A biological sample (droplets containing DNA and primer) (9) is prepared in a chemically inert liquid layer (8), is electrostatically transported by the action of an electrostatic transportation plate (1) with electrostatic transportation electrodes (2) and is heated at a predetermined position of the electrostatic transportation plate (1), thus carrying out PCR.

Claims

1. A method for carrying out PCR utilizing electrostatic transportation, comprising the steps of:

(a) preparing droplets of biological sample containing polymerase in a chemically inert liquid layer;

(b) electrostatically transporting the droplets of biological sample by the action of an electrostatic transportation plate with electrostatic transportation electrodes; and

(c) controlling temperatures at predetermined positions of the electrostatic transportation plate and carrying out polymerase chain reaction.

2. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, wherein the droplets of biological sample comprise droplets containing DNA and primer.

3. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, wherein the droplets of biological sample comprise droplets containing DNA, and droplets containing primer.

4. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, further comprising arranging the electrodes in matrix form and electrostatically transporting the droplets of biological AMENDED SHEET sample two-dimensionally.

5. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, further comprising applying voltage to heating electrodes and passing an electric current through the droplets of biological sample to thereby heat the droplets of biological sample.

6. The method for carrying out PCR utilizing electrostatic transportation according to claim 5, wherein high-frequency alternating voltages are applied as the voltage.

7. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, further comprising maintaining the chemically inert liquid layer at a first temperature with thermostatic heater.

8. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, further comprising heating the droplets of biological sample in multiple steps.

9. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, further comprising arranging the electrodes and regions to be heated in a cascade and treating the droplets of biological sample sequentially at specific time intervals.

19. The method for carrying out PCR utilizing electrostatic transportation according to claim 1, further comprising detecting and controlling the positions of the droplets of biological sample.

10. A device for carrying out PCR utilizing electrostatic transportation, comprising:

(a) a chemically inert liquid layer carrying droplets of biological sample containing polymerase;

(b) an electrostatic transportation plate covered with the chemically inert liquid layer and having electrodes with a water-repellent coating on a surface thereof

(c) means for electrostatically transporting droplets of biological sample by the action of the electrostatic transportation plate; and

(d) means for controlling the temperatures of the droplets of biological sample at predetermined positions of the electrostatic transportation plate.

11. The device for carrying out PCR utilizing electrostatic transportation according to claim 10, wherein the electrodes are arranged in matrix form.

12. The device for carrying out PCR utilizing electrostatic transportation according to claim 10, further comprising a sheet-like heater layer for thermostatically heating the chemically inert liquid layer.

13. The device for carrying out PCR utilizing electrostatic transportation according to claim 10, wherein the means for heating the droplets of biological sample is a heating plate for applying voltage to the droplets of biological sample.

14. The device for carrying out PCR utilizing electrostatic transportation according to claim 13, wherein the voltage is a high-frequency alternating voltage.

15. The device for carrying out PCR utilizing electrostatic transportation according to claim 10, wherein the means for heating the droplets of biological sample is a long heater layer.

20. The device for carrying out PCR utilizing electrostatic transportation according to claim 10 or 17, further comprising means including a computer, an image pickup device being connected to the computer, and a controller being connected to the computer, wherein the means is so configured as to monitor the motions of the droplets of biological sample with the image pickup device and to control the positions of the droplets of biological sample by the controller through the computer based on the monitoring.

16. A method for carrying out PCR utilizing electrostatic transportation, comprising the steps of:

(a) preparing droplets containing DNA and primer in a chemically inert liquid layer;

(b) electrostatically transporting the droplets containing DNA and primer by the action of an electrostatic transportation plate with electrostatic transportation electrodes;

(c) heating and thermally denaturing the DNA at a first position of the electrostatic transportation plate;

(d) annealing primer to the DNA, the primer being capable of reacting with the DNA; and

(e) elongating the annealed DNA.

23. The method for carrying out PCR utilizing electrostatic transportation according to claim 16, further comprising detecting and controlling the positions of the droplets containing DNA and a primer.

17. A device for carrying out PCR utilizing electrostatic transportation, comprising:

(a) an electrostatic transportation plate with electrodes, the electrodes each having a water-repellent coating on a surface thereof;

(b) a chemically inert liquid layer carrying droplets containing DNA and a primer;

(c) means for heating the droplets containing DNA and primer at predetermined positions;

(d) means for electrostatically transporting the droplets containing DNA and primer by the action of the electrostatic transportation plate;

(e) means for heating and thermally denaturing the DNA at a first position of the electrostatic transportation plate;

(f) means for annealing primer to the DNA, the primer being capable of reacting with the DNA; and

(g) means for elongating the annealed DNA.

18. The device for carrying out PCR utilizing electrostatic transportation according to claim 17, wherein the electrodes are arranged in matrix form.

21. A method for carrying out hybridization utilizing electrostatic transportation, comprising the steps of covering an electrostatic transportation electrode substrate with a chemically inert liquid, arranging an array of droplets each containing known single-stranded DNA and fluorescence reagent therein, electrostatically transporting droplets containing an unknown single-stranded DNA sample, combining the sample droplets with the respective droplets containing the known single-stranded DNA and the fluorescence reagent to thereby carry out hybridization, and detecting the hybridization based on the fact that the fluorescence reagent is intercalated between the known and unknown single-stranded DNAs when the two DNAs match with each other, and that fluorescence is emitted at a varying intensity depending on the degree of match between the two DNAs.

22. A device for carrying out hybridization utilizing electrostatic transportation, comprising:

(a) an electrostatic transportation electrode substrate being filled with a chemically inert liquid layer;

(b) droplets being arranged in an array on the electrostatic transportation electrode substrate and each comprising known single-stranded DNA and fluorescence reagent;

(c) droplets containing unknown single-stranded DNA sample to be transported by the action of electrodes of the electrostatic transportation electrode substrate; and

(d) means for treating the droplets containing the unknown single-stranded DNA sample with each of the droplets containing the known single-stranded DNAs and the fluorescence reagent and detecting the hybridization between the two DNAs.