Method and apparatus for amplification of nucleic acid sequences using immobilized DNA polymerase

• US 20040191830A1
• Filed: 04/29/2004
• Published: 09/30/2004
• Est. Priority Date: 10/30/2001
• Status: Active Grant

• Current Assignee: Ahram Biosystems, Inc.
• Original Assignee: Ahram Biosystems, Inc.
• Inventors: Kim, Jeong Hee, Hwang, Hyun Jin, Jeong, Kyunghoon
• Granted Patent: US 7,488,595 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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

Abstract

The present invention generally relates to methods and apparatuses for amplifying nucleic acid sequences using immobilized DNA polymerase. More particularly, it relates to methods and apparatuses useful for amplifying target nucleic acid sequences by forming a plurality of reaction regions in which polymerase chain reaction (PCR) can occur, positioning immobilized DNA polymerase in a specific reaction region, and circulating DNA through the reaction regions. The present invention provides those methods and apparatuses that allow simple separation and recovery of the DNA polymerase after the amplification, that can be operated not only with thermostable DNA polymerases but also with non-thermostable DNA polymerases, and that are simpler in their designs and processes so that they can be readily integrated into complex devices such as Lab-on-a-chip.

Claims

1. A method useful for amplifying at least one target DNA sequence using PCR, which method comprises:

a) a step of maintaining a first reaction region at a temperature range suitable for separating double stranded DNA molecules into single stranded DNA molecules,

a second reaction region at a temperature range suitable for hybridization of the single stranded DNA molecules with primers that are complementary to specific portions of the single stranded DNA molecules so as to form partially double stranded DNA molecules, and

a third reaction region at a temperature range suitable for DNA polymerization reaction of the primers in the partially double stranded DNA molecules so as to generate primer extension products;

b) a step of positioning an immobilized DNA polymerase in the third reaction region; and

c) a step of circulating the DNA molecules through the first, second, and third reaction regions to make amplification of the target DNA sequences occur,

wherein the step of circulating the DNA molecules is repeated at least once.

2. The method according to claim 1, wherein the immobilized DNA polymerase is an immobilized enzyme having a polymerase activity of replicating a sequence complementary to a template DNA, selected from the group consisting of E. Coli DNA polymerase I, Klenow fragment of E. Coli DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, and their homologs and derivatives.

3. The method according to claim 1, wherein the step of circulating the DNA molecules is induced by thermal convection generated by positioning a relatively high temperature region lower in height than a relatively low temperature region among the first, second, and third reaction regions.

4. The method according to claim 1, wherein the step of circulating the DNA molecules is induced by an electric field generating means.

5. The method according to claim 1, wherein the step of circulating the DNA molecules is induced by a pressure difference generating means.

6. The method according to claim 1, wherein the step of circulating the DNA molecules is induced by an agitating means.

7. An apparatus useful for amplifying at least one target DNA sequence using PCR, which apparatus comprises:

a) a reaction vessel;

b) means for maintaining a first reaction region of the reaction vessel at a temperature range suitable for separating double stranded DNA molecules into single stranded DNA molecules,

a second reaction region of the reaction vessel at a temperature range suitable for hybridization of the single stranded DNA molecules with primers that are complementary to specific portions of the single stranded DNA molecules so as to form partially double stranded DNA molecules, and

a third reaction region of the reaction vessel at a temperature range suitable for DNA polymerization reaction of the primers in the partially double stranded DNA molecules so as to generate primer extension products; and

c) means for circulating the DNA molecules through the first, second, and third reaction regions to make amplification of the target DNA sequences occur,

wherein an immobilized DNA polymerase is positioned in the third reaction region and the circulating means circulates the DNA molecules repeatedly at least once.

9. An apparatus according to any of claims 7 and 8, wherein the immobilized DNA polymerase is an immobilized enzyme having a polymerase activity of replicating a sequence complementary to a template DNA, selected from the group consisting of E. Coli DNA polymerase I, Klenow fragment of E. Coli DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, and their homologs and derivatives.

10. The apparatus according to claim 7, wherein the circulating means consists of an electric field generating means.

11. The apparatus according to claim 7, wherein the circulating means consists of a pressure difference generating means.

12. The apparatus according to claim 7, wherein the circulating means consists of an agitating means.

8. An apparatus useful for amplifying at least one target DNA sequence using PCR, which apparatus comprises:

a) a reaction vessel; and

b) means for maintaining a first reaction region of the reaction vessel at a temperature range suitable for separating double stranded DNA molecules into single stranded DNA molecules,

a second reaction region of the reaction vessel at a temperature range suitable for hybridization of the single stranded DNA molecules with primers that are complementary to specific portions of the single stranded DNA molecules so as to form partially double stranded DNA molecules, and

a third reaction region of the reaction vessel at a temperature range suitable for DNA polymerization reaction of the primers in the partially double stranded DNA molecules so as to generate primer extension products; and

wherein an immobilized DNA polymerase is positioned in the third reaction region and among the first, second, and third reaction regions, a relatively high temperature region is located lower in height than a relatively low temperature region so that the DNA molecules are circulated repeatedly at least once by thermal convection through the first, second, and third regions to make amplification of the target DNA sequences occur.