W0 02/060584
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`PCT/IB02/01048
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`30
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`sequences, followed by cycle sequencing (Happ et (1]., Vet Immunol Immunopathol, 69: 93-100,
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`(1999), incorporated herein by reference in its entirety); a degenerate PCR technique can be used as
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`a first step to identify and amplify the actual sequence when it is not completely known (Harwood ez‘
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`al., J Clin Microbiol 37: 3545—3555, (1999), incorporated herein by reference in its entirety);
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`random amplified DNA GQAPD) analysis, often useful as a exploratory approach (Speijer et al, J
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`Clin Microbiol 37: 3654-3 661 , (1999),
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`incorporated herein by reference in its entirety); and
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`arbitrarily primed PCR (AP—PCR), often useful
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`as
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`a preliminary step to find potential
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`polymorphisms (Jonas et al., J Clin Microbiol 38: 2284-2291, (2000),
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`incorporated herein by
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`reference in its entirety).
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`Although various devices have been designed for implementing chemical, biochemical, and
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`biological protocols comprising steps that include at least one thermal cycle, these devices have
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`serious drawbacks. Advantages provided by the present invention are apparent from comparing
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`other devices with the present invention.
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`In one type of known device, the biological sample is placed in a reservoir which is brought
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`successively to the required temperatures for obtaining the desired heat treatments. Certain devices
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`use a system of thermostatting or thermal regulation by Peltier effect. The heat response time of
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`these devices is on the order of 3°C per second (3°C/s). Although the method used by these devices
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`is simple to implement, the cycle times resulting therefrom are very long for carrying out the entire
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`protocol. This approach does not make it possible to increase the reaction throughput or yield.
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`Other techniques use a reservoir etched in a silicon substrate. The heating of the reservoir
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`is obtained by an electrical resistor which is formed by a platinum deposit. The cooling of the
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`reservoir is obtained by permanent conduction on a cold plate. To improve the heat response time
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`during the temperature cycles, the reservoir (or reaction chamber), is suspended by silicon beams
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`which are produced by chemical etching in the substrate. The heat response time is greater than
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`about ten degrees per second. The technique used is however relatively incompatible with the
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`integration of the protocols in a “laboratory on a chip”, 1'. e. in a microfluidic substrate where the
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`principle is to make the liquids circulate between various biological or biochemical reactions zones.
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`In this device, it is not possible to achieve continuous flow and thus to integrate a protocol adapted
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`to high throughput screening.
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`In another type of known device, microchannels which make it possible to circulate
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`biological fluids are micromachined on a substrate made of silicon, glass or polymer material.
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`These microchannels or capillaries make it possible to continually circulate the samples. The fluid
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`successively crosses zones at temperatures which are fixed according to the heat treatments
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`required. This solution leads to the production of microchannels in the form of coils. Figure 12
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`illustrates a device of this type, presenting a view from above of a substrate in which a
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`microchannel 1, represented figuratively, has been formed. The microchannel 1 forms a coil
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`between an inlet orifice 2 and an outlet orifice 3. The references 4, 5 and 6 represent zones which
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