Method and apparatus for amplification of nucleic acid sequences using immobilized DNA polymerase
First Claim
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1. 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, anda 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 exclusively positioned in the third reaction region of the reaction vessel and the circulating means circulates the DNA molecules repeatedly at least once; and
further wherein the apparatus comprises an opening adapted to receive the reaction vessel and having a closed end defined by the means for maintaining the first reaction region.
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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.
18 Citations
26 Claims
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1. An apparatus useful for amplifying at least one target DNA sequence using PCR, which apparatus comprises:
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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 exclusively positioned in the third reaction region of the reaction vessel and the circulating means circulates the DNA molecules repeatedly at least once; and
further wherein the apparatus comprises an opening adapted to receive the reaction vessel and having a closed end defined by the means for maintaining the first reaction region. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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2. An apparatus useful for amplifying at least one target DNA sequence using PCR, which apparatus comprises:
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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 of the reaction vessel 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; and
further wherein the apparatus comprises an opening adapted to receive the reaction vessel and having a closed end defined by the means for maintaining the first reaction region.
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Specification
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Current AssigneeAhram Biosystems, Inc.
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Original AssigneeAhram Biosystems, Inc.
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InventorsKim, Jeong Hee, Hwang, Hyun Jin, Jeong, Kyunghoon
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Primary Examiner(s)Horlick; Kenneth R.
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Assistant Examiner(s)Babic; Christopher M.
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Application NumberUS10/836,376Publication NumberTime in Patent Office1,748 DaysField of SearchNoneUS Class Current435/283.1CPC Class CodesB01L 2300/1838 using fluid heat transfer m...B01L 2300/1883 using thermal insulationB01L 2400/0442 thermal energy, e.g. vapori...B01L 2400/0445 Natural or forced convectionB01L 7/525 with physical movement of s...
