Method and apparatus for amplifying nucleic acids

• US 20050202489A1
• Filed: 03/03/2005
• Published: 09/15/2005
• Est. Priority Date: 03/12/2004
• Status: Active Grant

• Current Assignee: Samsung Electronics Co. Ltd.
• Original Assignee: Samsung Electronics Co. Ltd.
• Inventors: Namkoong, Kak, Lim, Geun-bae, Min, Jun-hong, Cho, Yoon-kyoung, Kim, Joon-ho
• Granted Patent: US 7,579,172 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 25/4337 Mixers with a diverging-converging cross-section
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 35/715 Feeding the components in several steps, e.g. successive steps
• CPC Class Codes: B01J 2219/00783 Laminate assemblies, i.e. the reactor comprising a stack of plates
• CPC Class Codes: B01J 2219/00824 Ceramic
• CPC Class Codes: B01J 2219/00831 Glass
• CPC Class Codes: B01J 2219/00833 Plastic
• CPC Class Codes: B01J 2219/00837 comprising coatings other than catalytically active coatings
• CPC Class Codes: B01J 2219/0086 Dimensions of the flow channels
• CPC Class Codes: B01J 2219/00873 Heat exchange
• CPC Class Codes: B01J 2219/00891 Feeding or evacuation
• CPC Class Codes: B01L 2300/0809 rectangular shaped
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 3/5027 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 B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: G01N 2035/00366 Several different temperatures used
• CPC Class Codes: G01N 2035/00514 Stationary mixing elements
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Abstract

Provided are a method and apparatus for amplifying nucleic acids. The method includes introducing into a reaction vessel via different inlet channels a reactant aqueous solution containing reactants for nucleic acid amplification and a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction, creating a plurality of reactant aqueous solution droplets surrounded by the fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel, and amplifying the nucleic acids in the reactant aqueous solution droplets. The apparatus includes a substrate, a reaction vessel formed inside of the substrate, at least one first inlet channel formed inside the substrate, connected to an end of the reaction vessel, and allowing introduction of a reactant aqueous solution containing reactants for nucleic acid amplification into the reaction vessel, a second inlet channel formed inside the substrate, connected to the end of the reaction vessel, and allowing introduction of a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction into the reaction vessel, and a heating unit installed on the substrate in such a way to thermally contact with the substrate and heating the substrate.

Claims

1. A method for amplifying nucleic acids, which comprises:

introducing into a reaction vessel via different inlet channels a reactant aqueous solution containing reactants for nucleic acid amplification and a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction;

creating a plurality of reactant aqueous solution droplets surrounded by the fluid by contacting the reactant aqueous solution with the fluid in the reaction vessel; and

amplifying the nucleic acids in the reactant aqueous solution droplets.

2. The method of claim 1, wherein the fluid is at least one selected from the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, and vegetable oil.

3. The method of claim 1, wherein the operation of amplifying the nucleic acids is performed by one selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and loop-mediated isothermal amplification (LAMP).

4. The method of claim 3, wherein the operation of amplifying the nucleic acids is performed by PCR.

5. The method of claim 4, wherein the operation of amplifying the nucleic acids is performed by PCR in which the entire surface of a substrate is repeatedly heated and cooled to a predetermined temperature.

6. The method of claim 4, wherein the operation of amplifying the nucleic acids is performed by continuous-flow PCR in which a substrate is heated to have at least two different temperature areas and the reactant aqueous solution droplets alternately and repeatedly pass through the different temperature areas.

7. The method of claim 6, wherein the substrate is heated to have at least two different temperature areas using a plurality of independently controllable heaters.

8. The method of claim 4, wherein the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel further comprises introducing into the reaction vessel at least one aqueous solution of a negative control and a positive control for quantitative assay and a standard sample aqueous solution via respective inlet channels different from the inlet channels for the introduction of the reactant aqueous solution and the fluid, and

the operation of creating the reactant aqueous solution droplets further comprises creating a plurality of control aqueous solution droplets and a plurality of standard sample aqueous solution droplets by contacting the at least one aqueous solution of the negative control and the positive control and the standard sample aqueous solution with the fluid in the reaction vessel.

9. The method of claim 8, wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, the standard sample aqueous solution is adjusted to at least two concentrations and then introduced into the reaction vessel.

10. The method of claim 4, wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, at least two reactant aqueous solutions containing at least two primers are introduced into the reaction vessel via respective inlet channels.

11. The method of claim 4, wherein in the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, a nucleic acid aqueous solution, a common component containing polymerase and dNTP, and a primer are introduced into respective inlet channels and mixed before being introduced into the reaction vessel to form the reactant aqueous solution.

12. The method of claim 1, wherein the reaction vessel comprises two primary reaction channels and a single secondary reaction channel connected to a terminal end of each of the two primary reaction channels, and the operation of introducing the reactant aqueous solution and the fluid into the reaction vessel, the operation of creating the reactant aqueous solution droplets, and the operation of amplifying the nucleic acids are performed in each of the two primary reaction channels, and

wherein after the operation of amplifying the nucleic acids, the method further comprises:

combining the reactant aqueous solution droplets in a starting end of the secondary reaction channel to create a plurality of combined reactant aqueous solution droplets in the secondary reaction channel; and

amplifying the nucleic acids in the combined reactant aqueous solution droplets.

13. The method of claim 1, wherein the reaction vessel comprises at least one reaction chamber, and

wherein after the operation of amplifying the nucleic acids in the reactant aqueous solution droplets, the method further comprises:

combining the reactant aqueous solution droplets in the reaction chamber; and

amplifying the nucleic acids in the combined reactant aqueous solution droplets.

14. The method of claim 13, wherein the operation of combining the reactant aqueous solution droplets is carried out by centrifugation.

15. An apparatus for amplifying nucleic acids, which comprises:

a substrate;

a reaction vessel formed inside of the substrate;

at least one first inlet channel formed inside the substrate, connected to an end of the reaction vessel, and allowing introduction of a reactant aqueous solution containing reactants for nucleic acid amplification into the reaction vessel;

a second inlet channel formed inside the substrate, connected to the end of the reaction vessel, and allowing introduction of a fluid that is phase-separated from the reactant aqueous solution and does not participate in amplification reaction into the reaction vessel; and

a heating unit installed on the substrate and heating the substrate,

the reactant aqueous solution contacting with the fluid to create a plurality of reactant aqueous solution droplets surrounded by the fluid in the reaction vessel and the nucleic acids being amplified in the reactant aqueous solution droplets.

16. The apparatus of claim 15, wherein the fluid is at least one selected from the group consisting of silicon oil, mineral oil, perfluorinated oil, hydrocarbon oil, and vegetable oil.

17. The apparatus of claim 15, wherein the substrate has a hydrophobic surface property.

18. The apparatus of claim 17, wherein the substrate is made of one material selected from the group consisting of poly(dimethylsiloxane) (PDMS), silicon, silicon dioxide, plastic, and glass.

19. The apparatus of claim 15, wherein the substrate comprises a lower substrate and an upper substrate, and the reaction vessel, the first inlet channel, and the second inlet channel are formed between the lower substrate and the upper substrate.

20. The apparatus of claim 15, wherein the nucleic acids are amplified in the reactant aqueous solution droplets by one selected from the group consisting of PCR, LCR, SDA, NASBA, TMA, and LAMP.

21. The apparatus of claim 20, wherein the nucleic acids are amplified by PCR in the reactant aqueous solution droplets.

22. The apparatus of claim 21, wherein the reaction vessel is a serpentine reaction channel, the first inlet channel and the second inlet channel are connected to a staring end of the reaction channel, and an outlet port for releasing the fluid and an amplification product is connected to a terminal end of the reaction channel.

23. The apparatus of claim 22, wherein the heating unit is installed at a lower surface of the substrate and comprises at least two heaters for heating the substrate to allow the substrate to have at least two different temperature areas.

24. The apparatus of claim 22, wherein the heating unit comprises a plurality of independently controllable heaters installed in parallel at a lower surface of the substrate to heat the substrate so that the substrate has at least two temperature areas.

25. The apparatus of claim 23, wherein the reaction channel alternately and repeatedly passes through the temperature areas.

26. The apparatus of claim 22, wherein the second inlet channel linearly extends from the starting end of the reaction channel and the first inlet channel is formed perpendicularly to the second inlet channel.

27. The apparatus of claim 22, further comprising:

a third inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of at least one selected from a negative control aqueous solution and a positive control aqueous solution for quantitative assay into the reaction channel; and

a fourth inlet channel formed inside the substrate, connected to the end of the reaction channel, and allowing introduction of a standard sample aqueous solution for quantitative assay into the reaction channel.

28. The apparatus of claim 27, wherein the fourth inlet channel is connected to at least two standard sample inlet channels for introduction of standard samples and a distilled water inlet channel for diluting the standard samples to a predetermined concentration.

29. The apparatus of claim 28, wherein the fourth inlet channel has a serpentine shape.

30. The apparatus of claim 22, wherein the apparatus comprises at least two first inlet channels and the first inlet channels are correspondingly connected to at least two primer inlet channels for introduction of at least two different primers into the first inlet channels.

31. The apparatus of claim 30, wherein each of the first inlet channels has a serpentine shape.

32. The apparatus of claim 22, wherein the first inlet channel is connected to a primer inlet channel for introduction of a primer into the first inlet channel and a common component inlet channel for introduction of a common component comprising polymerase and dNTP into the first inlet channel.

33. The apparatus of claim 32, wherein a portion of a starting end side of the reaction channel has a serpentine shape.

34. The apparatus of claim 15, wherein the reaction vessel comprises two serpentine primary reaction channels and a single serpentine secondary reaction channel connected to a terminal end of each of the two primary reaction channels, and the first inlet channel and the second inlet channel are connected to starting ends of the two primary reaction channels.

35. The apparatus of claim 34, wherein the heating unit comprises a plurality of independently controllable heaters installed at a lower surface of the substrate to provide two temperature areas for each of the two primary reaction channels and the single secondary reaction channel.

36. The apparatus of claim 15, wherein the reaction vessel is a reaction chamber, the first inlet channel and the second inlet channel are connected to an end of the reaction chamber, and an outlet channel for releasing the fluid and an amplification product is connected to the other end of the reaction chamber.

37. The apparatus of claim 36, wherein the apparatus comprises a plurality of reaction chambers, first inlet channels, second inlet channels, and outlet channels.

38. The apparatus of claim 36, wherein the heating unit comprises a single heater installed at a lower surface of the substrate and heating the entire surface of the substrate to a predetermined temperature.