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1. (WO1991007504) IMPROVED DNA POLYMERISATION DEVICE
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IMPROVED DNA POLYMERISATION DEVICE
Field of the Invention
The present invention relates to a device for use in DNA polymerisation.
Background of the Invention
In a number of applications, such as gene analysis and DNA fingerprinting, it is often necessary to multiply the amount of DNA present a sample. A DNA segment of up to approximately six thousand base pairs in length may be amplified exponentially starting from as little as a single gene copy by means of polymerised chain reaction.
In this technique a denatured DNA sample is incubated with two oligonucleotide primers that direct the DNA polymerase-dependent synthesis of complimentary strains. Multiple cycles of synthesis each afford an approximate doubling of the amount of target sequence. Each cycle is controlled by simply varying the temperature to permit denaturation of the DNA strands, annealing of the primers, and synthesis of new DNA strands. The use of a
thermostable DNA polymerase obviates the necessity of adding new enzyme for each cycle, thus enabling fully automated DNA amplification. Twenty-five amplification cycles increase the amount of target sequence by
approximately 10^-fold. For the purposes of gene
analysis the polymerase chain reaction technique offers the advantage of an increased signal intensity in
subsequent assays. More detailed information regarding the polymerase chain reaction can be found in "PCR
Protocols - A Guide to Methods and Applications" Eds. M.A. Innis, D.H. Gelfard, J.J. Sainskey, T.J. White, Academic Press. Inc. San Diego 1990" the disclosure of which is incorporated herein by reference.
Devices for use in DNA polymerisation typically consist of a heat conductive material provided with channels adapted to receive vessels in which the reaction is to take place, typically Eppendorf tubes. The heat conductive material is then provided with heating/cooling means. One of the main difficulties encountered in the use of such devices has been the achievement of relatively rapid cooling of the DNA reaction mixture. The most common solution to this problem has been to use Peltier effect heat pumps to effect both the heating and the cooling of the reaction mixture. However, the continual cycling between heating and cooling typically leads to the Peltier effect heat pumps failing.
Summary of the Invention
The present inventor has developed a novel device which enables rapid heating and cooling of the DNA
reaction mix whilst being relatively robust and economic to produce.
Accordingly, in a first aspect the present invention consists in a device for use in DNA polymerisation, the device comprising an element adapted to receive tubes in which the DNA polymerisation reaction is to take place and being provided with heating means adapted to heat the element, a heat sink adapted to remove heat from the element and movement means enabling the movement of the element and the heat sink into and out of contact with each other.
In a second aspect the present invention consists in a method of DNA amplification using polymerase chain reaction, the method being characterised in that a reaction mixture comprising DNA, suitable thermoscable DNA poly erases and oligonucleotide primers in a vessel is placed in an element adapted to receive the vessel, the element being provided with heating means, heating the reaction mixture by actuation of the heating means, stopping the heating means, cooling the reaction mixture by bringing a heat sink into contact with the element, moving the heat sink away from the element and cyclically repeating the heating and cooling steps.
In a preferred embodiment of the present invention the element remains stationary and the heat sink is moved into and out of contact with the element.
In a further preferred embodiment of the present invention the mass of the heat sink is substantially greater than the mass of the element. It is presently preferred that the mass of the heat sink is two to three times the mass of the element.
In yet a further preferred embodiment of the present invention the device is provided with means for cooling the heat sink. This cooling means may consist of a fan directed on the heat sink, Peltier effect coolers provided within the heat sink or other forms of cooling readily known in the art.
In yet another preferred embodiment of the present invention the movement of the element and/or the heat sink into and out of contact with each other is automated.
Preferably, in operation, temperature sensing means is provided in the DNA reaction mixture. The temperature sensing means is preferably connected to control means which controls the movement of the element and/or heat sink into and out of contact with each other. It is preferred that this automation is computer controlled. Detailed Description of the Invention
In order that the nature of the present invention may be more clearly understood, a preferred form thereof will now be described with reference to the accompanying drawing, in which is shown a schematic representation of a preferred embodiment of the device of the present
invention.
The device 10 for use in DNA polymerisation comprises an element 12, a heat sink 14 and movement means 16. The element 12 and heat sink 14 are made of material which is a good thermal conductor, such as metal.

The element 12 is provided with wells 18 adapted to receive tubes in which the DNA polymerisation reaction is to take place. The element 12 is also provided with heat electric heating elements shown generally as 20.
The heat sink 14 is provided with cooling fins 22 to enable a greater dissipation of heat from the heat sink. The cooling fins 22 are positioned above a fan 25 arranged such as to blow air onto the cooling fins 22 of the heat sink 14.
The heat sink 14 is also provided with Peltier effect coolers shown generally as 24.
The movement means 16 enables the movement of the heat sink 14 into and αμt of contact with the element 12. The movement means 16 consists of arm 26 connected at. end 28 to heat sink 14 and at end 30 to actuator 32. The arm 26 is pivoted about point 34. Actuation of
actuator 32 will result in the heat sink 14 being brought into contact with the element 12.
In order that the nature of the operation of the device of the present invention may be more clearly understood the operation of the device shown in the accompanying drawing will now be described.
A reaction mixture of DNA, thermostable DNA
polymerase and oligonucleotide primers in tubes are placed in wells 18 of element 12. The electric heating
elements 20 are then actuated to raise the temperature of the element 12 and consequently the temperature of the reaction mix in the tubes in wells 18. The temperature is then maintained at a predetermined level, typically 93°C for a predetermined period of time, typically one and a half minutes. At the end of the heating period the electric heater elements 20 are turned off and actuator 32 actuated resulting in bringing heat sink 14 into contact with element 12. As heat sink 14 is at a substantially lower temperature than element 12 heat is lost from element 12 to heat sink 14. The reaction mixture is cooled to a predetermined temperature, typically 60°C and held at this temperature for a predetermined period of time, typically one and a half minutes.
Typically, given the substantially greater mass of the heat sink 14 in comparison to element 12, the
adsorption of heat from element 12 by heat sink 14 results in little raising of the temperature of heat sink 14. In addition, heat is rapidly lost from the heat sink 14 due to the action of fan 25 directing air on cooling fins 22. In addition, the temperature of the heat sink 14 may .also be lowered by Peltier coolers 24.
At the end of the cooling step the actuator 32 is once again actuated and heat sink 14 is moved out of contact with element 12. At this time the electric heater elements are again actuated and the reaction mixture heated once again.
These steps are then cyclically repeated until the desired number of amplification steps are achieved.
In use the device of the present invention has been shown to be capable of achieving the drop in temperature from 93°C to 60° in approximately thirty seconds
without the use of the Peltier effect coolers. As will be appreciated by persons skilled in the art this is a substantial improvement over prior art devices.
As would be appreciated by persons skilled in the art the cyclic nature of the operation of the device of the present invention lends itself to computer automation. In such a situation the temperature of the element 12 and the DNA reaction mixture is monitored and the electric heating elements 20 cyclically actuated and the heat sink 14 cyclically brought into contact with the element 12 by means of movement means 16 to achieve cooling of the element 12 and consequently the reaction mixture.
As would be appreciated by persons skilled in the art such DNA amplification procedures are typically run over an extended period of time to provide the required number of amplification cycles. If desired at the end of the required number of cycles, the computer can be programmed such that the heat sink 14 is brought into contact with the element 12 and the Peltier coolers 26 actuated to maintain the DNA reaction mixture at a predetermined temperature such as 4°C. Any risk of substantial
denaturation of the amplified DNA would therefore be reduced.
As will be appreciated by persons skilled in the art the device of the present invention provides a relatively robust and simple alternative to devices of the prior art whilst providing a rapid means of cooling the DNA reaction mixture.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the
invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.