Method and apparatus for continuous amplification of DNA

• US 20040076952A1
• Filed: 10/19/2002
• Published: 04/22/2004
• Est. Priority Date: 07/12/2001
• Status: Abandoned Application

• Current Assignee: Toshio Kawai
• Original Assignee: Toshio Kawai
• Inventors: Kawai, Toshio
• Application Number:
  US10/065,456
• Publication Number:
  US 20040076952A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2300/185 using a liquid as fluid
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 7/02 Water baths; Sand baths; Air baths
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones

Abstract

Continuous DNA-amplification method and apparatus. Reaction mixture containing reagent solution and DNA fragments serving as templates is continuously temperature-processed by heat-exchange fluids whose temperatures differ, enabling successive DNA amplification reactions to be carried out efficaciously in large volume. Apparatus has a reaction-mixture tank, isothermal denaturing, annealing and elongation tanks, a recirculation path that circuits the isothermal tanks, and a pump that works to feed reaction mixture within the recirculation path unidirectionally through it. The reaction mixture, transferred from tank to tank by the pump, is maintained at prescribed reaction temperatures in the isothermal tanks. Heat-exchange efficiency is improved compared with batch systems, and the reaction mixture—uniformly, swiftly put into prescribed temperature states—can be amplification-reacted under ideal conditions, enabling quantum improvement in the amplification efficiency. Amplification reactions are carried out by circuit-feeding the reaction mixture, which serves to establish per-cycle DNA amplification quantity at large volumes.

Claims

1. A method for continuous amplification of DNA, the method repeating DNA amplification reactions continuously by:

with a recirculation path and a pump furnished in the recirculation path, feeding unidirectionally through the recirculation path a reaction mixture containing DNA fragments and a reagent solution, held in a reaction-mixture tank;

sending the reaction mixture within the recirculation path by way of, in order, a denaturing isothermal tank wherein a temperature for dissolving apart the DNA'"'"'s double strands is maintained, an annealing isothermal tank wherein a temperature at which primers contained in the reagent solution anneal to the DNA fragments is maintained, and an elongation isothermal tank wherein a temperature at which complementary chains are extended continuously onto the primers is maintained; and

with heat-exchange fluids within the isothermal tanks, maintaining the reaction mixture for predetermined times at predetermined temperatures, and subsequently recirculating the reaction mixture into the reaction-mixture tank.

2. The DNA continuous amplification method set forth in claim 1, wherein the amplification reactions are performed by making time for heat-exchange in the denaturing isothermal tank a reference time, and setting individual heat-exchange times in the annealing isothermal tank and the elongation isothermal tank as multiples of the reference time.

3. A method for continuous amplification of DNA, the method repeating DNA amplification reactions continuously by:

circuit-feeding unidirectionally through an endless recirculation path, using a pump provided therein, a reaction mixture containing DNA fragments and a reagent solution, held within the recirculation path;

circulating the reaction mixture within the recirculation path by way of, in order, a denaturing isothermal tank wherein a temperature for dissolving apart the DNA'"'"'s double strands is maintained, an annealing isothermal tank wherein a temperature at which primers contained in the reagent solution anneal to the DNA fragments is maintained, and an elongation isothermal tank wherein a temperature at which complementary chains are extended continuously onto the primers is maintained; and

with heat-exchange fluids within the isothermal tanks, maintaining the reaction mixture for predetermined times at predetermined temperatures.

4. An apparatus for continuous amplification of DNA, comprising:

a reaction-mixture tank for holding a reaction mixture containing DNA fragments and a reagent solution;

a denaturing isothermal tank for holding a heat-exchange fluid adjusted to a temperature for dissolving apart the DNA'"'"'s double strands;

an annealing isothermal tank for holding a heat-exchange fluid adjusted to a temperature at which primers contained in the reagent solution anneal to the DNA fragments;

an elongation isothermal tank for holding a heat-exchange fluid adjusted to a temperature at which complementary chains are extended continuously onto the primers;

a recirculation-path system through which the reaction mixture in the reaction-mixture tank is fed and guided, the recirculation-path system being arranged such that it circuits from the reaction-mixture tank and goes by way of the denaturing isothermal tank, the annealing isothermal tank, and the elongation isothermal tank back to the reaction-mixture tank; and

a pump working to feed the reaction mixture in said recirculation-path system unidirectionally through it;

wherein

the apparatus is configured such that the reaction mixture in said recirculation-path system is for timed intervals maintained at prescribed temperatures determined by the heat-exchange fluids in the isothermal tanks.

5. The DNA continuous amplification apparatus set forth in claim 4, wherein said denaturing isothermal tank, said annealing isothermal tank, and said elongation isothermal tank each respectively include:

a container body for holding the heat-exchange fluid;

a heat source for heating the heat-exchange fluid to, and retaining it at, the prescribed temperatures; and

a stirring device for stirring the heat-exchange fluid.

6. The DNA continuous amplification apparatus set forth in claim 4, wherein said denaturing isothermal tank, said annealing isothermal tank, and said elongation isothermal tank each respectively include:

a container body for holding the heat-exchange fluid;

a stirring device for stirring the heat-exchange fluid; and

a heating device containing a pump for circuit-feeding the heat-exchange fluid in between the container body and the heating device, and a heat source for heating the heat-exchange fluid to and retaining it at prescribed temperatures, wherein said heating device supplies the heat-exchange fluid to said container body.

7. The DNA continuous amplification apparatus set forth in claim 4, wherein a plurality of said recirculation-path systems in parallel is provided along with said pump between the reaction-mixture tank and said container bodies.

8. The DNA continuous amplification apparatus set forth in claim 5, wherein a plurality of said recirculation-path systems in parallel is provided along with said pump between the reaction-mixture tank and said container bodies.

9. The DNA continuous amplification apparatus set forth in claim 6, wherein a plurality of said recirculation-path systems in parallel is provided along with said pump between the reaction-mixture tank and said container bodies.

10. The DNA continuous amplification apparatus set forth in claim 4, further comprising:

a plurality of sets of said denaturing isothermal tank, said annealing isothermal tank, and said elongation isothermal tank; and

a plurality of said recirculation-path systems in parallel, provided along with said pump between the reaction-mixture tanks and the isothermal tanks of said plurality of sets.

11. The DNA continuous amplification apparatus set forth in claim 5, further comprising:

a plurality of sets of said denaturing isothermal tank, said annealing isothermal tank, and said elongation isothermal tank; and

a plurality of said recirculation-path systems in parallel, provided along with said pump between the reaction-mixture tanks and the isothermal tanks of said plurality of sets.

12. The DNA continuous amplification apparatus set forth in claim 6, further comprising:

a plurality of sets of said denaturing isothermal tank, said annealing isothermal tank, and said elongation isothermal tank; and

a plurality of said recirculation-path systems in parallel, provided along with said pump between the reaction-mixture tanks and the isothermal tanks of said plurality of sets.

13. The DNA continuous amplification apparatus set forth in claim 4, further comprising a coiled heat-exchange path immersed into each of said isothermal tanks in sections along the way of said recirculation-path system.

14. The DNA continuous amplification apparatus set forth in claim 5, further comprising a coiled heat-exchange path immersed into each of said isothermal tanks in sections along the way of said recirculation-path system.

15. The DNA continuous amplification apparatus set forth in claim 6, further comprising a coiled heat-exchange path immersed into each of said isothermal tanks in sections along the way of said recirculation-path system.

16. The DNA continuous amplification apparatus set forth in claim 7, further comprising a coiled heat-exchange path immersed into each of said isothermal tanks in sections along the way of said recirculation-path system.

17. The DNA continuous amplification apparatus set forth in claim 10, further comprising a coiled heat-exchange path immersed into each of said isothermal tanks in sections along the way of said recirculation-path system.