Method and apparatus for amplification of nucleic acid sequences by using thermal convection

• US 8,053,215 B2
• Filed: 10/30/2009
• Issued: 11/08/2011
• Est. Priority Date: 09/15/2001
• Status: Expired due to Fees

• Current Assignee: Ahram Biosystems, Inc.
• Original Assignee: Ahram Biosystems, Inc.
• Inventors: Kim, Jeong Hee, Hwang, Hyun Jin, Jeong, Kyunghoon
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US12/610,245
• Publication Number:
  US 20100285536A1
• Time in Patent Office: 739 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: B01L 2300/1838 using fluid heat transfer medium
• CPC Class Codes: B01L 2300/1883 using thermal insulation
• CPC Class Codes: B01L 2400/0442 thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 2400/0445 Natural or forced convection
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present invention provides a nucleic acid sequence amplification method and apparatuses thereof that are simple in the design and easy to miniaturize and integrate into complex apparatuses, with capability of using DNA polymerases that are not thermostable. In the present invention, a plurality of heat sources are combined to supply heat to, or remove heat from specific regions of the sample such that a specific spatial temperature distribution is maintained inside the sample by locating a relatively high temperature region lower in height than a relatively low temperature region.

Claims

1. A nucleic acid sequence amplification method using polymerase chain reaction (PCR), which method comprises:

a step of injecting into a reaction vessel a sample comprising a template DNA having target nucleic acid sequences to be amplified and DNA polymerase, and at least two oligonucleotide primers complementary to the 3′

terminus of each of the target nucleic acid sequences; and

a step of maintaining a spatial temperature distribution in the sample by contacting thermally with the sample a plurality of heat sources which supply heat to, or remove heat from regions of the sample such that a relatively high temperature region is located lower in height than a relatively low temperature region,

wherein the spatial temperature distribution comprises spatial regions fulfilling temperature conditions suitable for (i) a denaturation step in which double stranded DNAs become separated to single stranded DNAs, (ii) an annealing step in which the single stranded DNAs formed in the denaturation step hybridize to the primers to form DNA-primer complexes, or (iii) a polymerization step in which the primers in the DNA-primer complexes are extended by the polymerization reaction,

and wherein the spatial temperature distribution is a temperature distribution that induces circulation of the sample by thermal convection so that the denaturation, annealing, and polymerization steps occur sequentially and repeatedly inside the sample.

2. The nucleic acid sequence amplification method of claim 1, wherein at least one of the heat sources comprises a thermally conductive solid in thermal contact with a specific region of the reaction vessel or the sample;

and a heating unit that supplies heat to the thermally conductive solid, a cooling unit that removes heat from the thermally conductive solid, or a combination of the heating unit and the cooling unit.

3. The nucleic acid sequence amplification method of claim 1, wherein at least one of the heat sources comprises a liquid in thermal contact with a specific region of the reaction vessel;

a receptor in which the liquid is to be contained; and

a heating unit that supplies heat to the liquid, a cooling unit that removes heat from the liquid, or a combination of the heating unit and the cooling unit.

4. The nucleic acid sequence amplification method of claim 3, wherein at least one of the heat sources further comprises a circulation unit that circulates the liquid around the reaction vessel.

5. The nucleic acid sequence amplification method of claim 1, wherein at least one of the heat sources comprises a gas in thermal contact with a specific region of the reaction vessel;

a heating unit that supplies heat to the gas, a cooling unit that removes heat from the gas, or a combination of the heating unit and the cooling unit; and

a circulation unit that circulates the gas around the reaction vessel.

6. The nucleic acid sequence amplification method of claim 1, wherein at least one of the heat sources is an infrared radiation generating unit that supplies heat directly to the sample.

7. The nucleic acid sequence amplification method of claim 1, which method uses a means for insulating heat transfer between the heating sources.

8. The nucleic acid sequence amplification method of claim 1, wherein the plurality of the heat sources comprises a first thermally conductive solid that is in thermal contact with a lower portion of the reaction vessel and a second thermally conductive solid that is in thermal contact with an upper portion of the reaction vessel.

9. The nucleic acid sequence amplification method of claim 8, wherein the plurality of the heat sources further comprises a third thermally conductive solid that is in thermal contact with an intermediate portion of the reaction vessel in between the upper and lower portions.

10. The method of claim 1, wherein the method further comprises the step of denaturing the nucleic acid sequences to be amplified in the presence of the sample circulation and thermal convection.

11. The method of claim 10, wherein the denaturation step occurs at a temperature of 90 to 94°

C. or about 96°

C.

12. The method of claim 1, wherein the method further comprises the step of annealing the nucleic acid sequences to be amplified in the presence of the sample circulation and thermal convection.

13. The method of claim 12, wherein the annealing step occurs at a temperature of 35 to 65°

C.

14. The method of claim 1, wherein the method further comprises the step of amplifying the nucleic acid sequences to be amplified in the presence of the sample circulation and thermal convection.

15. The method of claim 14, wherein the annealing step occurs at a temperature of 72°

C.

16. The method of claim 1, wherein the DNA Polymerase is a Taq Polymerase.

17. The method of claim 1, wherein the DNA Polymerase is not thermostable.

18. The method of claim 16, wherein the DNA Polymerase is a Klenow fragment or a T7 DNA Polymerase.

19. The method of claim 1, wherein the DNA Polymerase is immobilized on a support.