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`Example 4
`
`Identifying new polymorphisms using arbitrarily primed PCR (AP-PCB)
`
`The method of arbitrarily primed PCR lAP-PCR) is often useful as a preliminary step to find potential
`
`polymorphisms. Jonas er al. (J. Clin. Microbiol. 38: 2284-2291 (2000)). AP-PCR can be carried out as follows: a
`
`reaction volume of 2.5 ul comprising 0.1 ul of cell lysate; 10 mM HCl, 3 mM MgCl2, 50 mM KCI, 0.2 mM dNTPs, 1
`
`units Taq DNA polymerase, with M13 primer fluorescently labelled with Cy-5, at pH 9.0.
`
`In one embodiment, the
`
`reaction mixture is made prior to introducing it into a channel of the present invention: in another embodiment,
`
`components of the reaction mixture are introduced directly into the device. Continuously flowing through temperature
`
`regulated zones, the reaction mixture is exposed to 45 cycles of 95°C for 60 s, 36°C for 60 s, and 72°C for 120 5.
`
`Reaction products may be sequenced to identify polymorphisms in sim in the device as described above, or
`
`alternately, removed for analysis.
`
`Example 5
`
`Genotyping by the TaqMan” PCR method
`
`A homogeneous phase protocol for TaqManTM PCR can be carried out in accordance with the present
`
`invention using a microfluidic substrate essentially as described in Example 2.
`
`TaqManTM is carried out in parallel in several (for example 3) channels. The volume of one PCR reaction is
`
`0.6m. Alternatively, the product of a single PCR reaction is detected.
`
`The microfluidic substrate is siliconized just before the substrate is used. Before using the substrate, all the
`
`channels are filled with previously degassed and filtered water. A high flow rate of the order of 25 ullmin is applied
`
`for 15 minutes to remove all the air bubbles present in the circuits. A previously degassed and filtered solution of
`
`10 mM Tris-HCl and 50 mM KCI pH 8.3 is then injected at a flow rate of 5 pllmin for 15 min, taking care not to form
`
`air bubbles.
`
`Each DNA sample is diluted in a previously degassed and filtered 1 mM Tris-HCl, 0.1 mM EDTA pH 8.3
`
`buffer, to obtain 30 pl of a final solution at 2nglul in DNA (enough to perform 50 different molecular beacon
`
`gentyping reactions with the same sample). 0ne DNA preparation is used for all three channels. Each PCR reaction
`
`uses 0.6 pl of this solution. This solution is placed in a device for injecting the samples in parallel and in series.
`
`A TaqManTM PCR reagent mixture comprising one pair of PCR primers and two allele specific molecular
`
`probes (one for each allele of the target nucleotide) labelled with a reporter and quencher dye is used, and total of 6 pl
`
`of a solution is prepared: 0.6 pM of each unpurified PCR primer. 20 mM Tris-HCI and 100 mM KCI pH 8.3 (or TaqMan
`
`buffer A, PE Applied Biosystems), previously degassed and filtered, 4 mM MgCl2, 0.2 mM of custom labelled probe
`
`and of each dNTP (dATP, dCTP, dGTP, dTTP), and 0.5U of AmpliTaq Gold. Each PCR reaction used 0.6m of this
`
`solution. These solutions were placed in the device for injecting the PCR primers in series; and were stored at 4°C. Up
`
`to 50 different reaction mix corresponding to 50 different polymorphic sites, can be prepared by changing the PCR
`
`primers probes and TaqMan probes. Care will be taken to have PCR products with equivalent size and TaqMan probes
`
`with similar Tms in order to keep the same cycling temperatures for all the different reactions. The different reaction
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`mix are injected one after the other in the device, however when injected the mix is injected in all the channels in
`
`parallel.
`
`The flow rates in the substrate is 8.2pllhour, such that ~ 35 cycles are carried out on 2cm of the PCR
`
`temperature regulated cycling zone.
`
`This reaction mixture can be treated before injection into a device at 94°C for 10 minutes if necessary to
`
`activate the polymerase. It is run through a zone which is cycled, in which a cycle consists of a step of 20 sec. at
`
`94°C, then 20 sec. at 55°C and 20 sec. at 72°C, for a duration corresponding to 35 cycles.
`
`Once the PCR reaction flows out of the PCR temperature regulated zone, the PCR is completed, the reaction
`
`can then pass a thermostated detection zone consisting of a fluorimeter adapted to the TaqMan probes wave length
`
`10
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`and continuous flow detection. In an other embodiment the PCR reactions can be collected from the oulet basin and
`
`analyzed_on an ABI Prism 7700 Sequence Detection System according to the manufacturers instructions.
`
`Example 6
`
`Genotyping using molecular beacons
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`25
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`30
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`A homogeneous phase protocol using molecular beacons can be carried out in accordance with the present
`
`invention using a microfluidic substrate essentially as described in Example 2.
`
`The assay is carried out in parallel in several (for example 3) channels. The volume of one PCR reaction is
`
`l-lel- Alternatively, the product of a single PCR reaction is detected.
`
`The microfluidic substrate is siliconized just before the substrate is used. Before using the substrate, all the
`
`channels are filled with previously degassed and filtered water. A high flow rate of the order of 25 pllmin is applied
`
`for 15 minutes to remove all the air bubbles present in the circuits. A previously degassed and filtered solution of
`
`10 mM Tris-H01 and 50 mM KCI pH 8.3 is then injected at a flow rate of 5 pllmin for 15 min, taking care not to form
`
`air bubbles.
`
`Each DNA sample is diluted in a previously degassed and filtered 1 mM Tris-HUI, 0.1 mM EDTA pH 8.3
`
`buffer, to obtain 30 pl of a final solution at 2nglp.l in DNA (enough to perform 50 different molecular beacon
`
`gentyping reactions with the same sample). 0ne DNA preparation is used for all three channels. Each PCR reaction
`
`uses 0.6 pl of this solution. This solution is placed in a device for injecting the samples in parallel and in series.
`
`A PCR reagent mixture comprising one pair of PCR primers and two allele specific molecular beacon probes
`
`(one for each allele of the target nucleotide) labelled with a reporter (FAM) and quencher dye (TAMRA) is used, and a
`
`total of 6 pl of a solution is prepared: 0.6 pM of each unpurified PCR primer, 20 mM Tris-HCI and 100 mM KCI
`
`pH 8.3 previously degassed and filtered, 4 mM MgCl2, 0.2 mM of molecular beacon probe and of each dNTP (dATP,
`
`dCTP, dGTP, dTTP), and 0.50 of AmpliTaq Gold. Each PCR reaction used 0.6 pl of this solution. These solutions were
`
`placed in the device for injecting the PCR primers in series; and were stored at 4°C. Up to 50 different reaction mix
`
`corresponding to 50 different polymorphic sites, can be prepared by changing the PCR primers probes and molecular
`
`beacons probes. Preferably, the PCR products have equivalent sizes and the molecular beacon probes have similar
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`Tms in order to keep the same cycling temperatures for all the different reactions. The different reaction mix are
`
`injected one after the other in the device, however when injected the mix is injected in all the channels in parallel.
`
`The flow rates in the substrate is 8.2ullhour, such that ~ 35 cycles are carried out on 2cm of the PCR PCR
`
`temperature regulated cycling zone.
`
`This reaction mixture can be treated before injection into a device at 94°C for 10 minutes if necessary to
`
`activate the polymerase. II is run through a zone which is cycled, in which a cycle consists of a step of 20 sec. at
`
`94°C, then 20 sec. at 55°C and 20 sec. at 72°C, for a duration corresponding to 35 cycles.
`
`Once the PCR reaction flows out of the PCR temperature regulated zone, the PCR is completed, the reaction
`
`can then pass a thermostated detection zone consisting of a fluorimeter adapted to the molecular beacon wave length
`
`10
`
`and continuous flow detection. In an other embodiment the PCR reactions can be collected from the oulet basin and
`
`analyzed on an ABI Prism 7700 Sequence Detection System according to the manufacturers instructions.
`
`Although this invention has been described in terms of certain preferred embodiments, other embodiments
`
`which will be apparent to those of ordinary skill in the art in view of the disclosure herein are also within the scope of this
`
`invention. Accordingly, the scope of the invention is intended to be defined only by reference to the appended claims. All
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`documents cited herein are incorporated herein by reference in their entirety.
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`WHAT IS CLAIMED IS:
`
`1.
`
`A device comprising:
`
`a microfluidic substrate comprising at least one pathway for sample flow; and
`
`at least one thermal transfer member which is capable of cycling between at least two
`
`temperatures, said at least one thermal transfer member being adapted to bring at least a portion of said
`
`sample pathway to said at least two temperatures while a sample is continuously flowing along said at least
`
`a portion of said sample pathway.
`
`2.
`
`The device of Claim 1, further comprising a force supplying member operably linked to said at least one
`
`pathway for sample flow wherein said force supplying member applies a force to said sample such that said sample
`
`10
`
`travels along said at least one pathway.
`
`3.
`
`The device of Claim 2, further comprising a sample supplier which supplies a sample to said at least one
`
`pathway.
`
`4.
`
`The device of Claim 3, further comprising at least one inlet basin positioned at a first end of said at
`
`least one pathway such that said sample supplier supplies said sample to said inlet basin and said sample travels from
`
`15
`
`said inlet basin to said at least one pathway.
`
`5.
`
`The device of Claim 4, further comprising at least one outlet basin positioned at a second end of said
`
`pathway.
`
`6.
`
`The device of Claim 5, further comprising at least one reagent supplier positioned between said inlet
`
`basin and said outlet basin.
`
`20
`
`7.
`
`8.
`
`9.
`
`The device of Claim 6, wherein said device comprises a plurality of said pathways.
`
`The device of Claim 7, wherein said pathways comprise channels arranged in parallel.
`
`The device of Claim 8, wherein the force generated by said force supplying member is pressure.
`
`10. The device of Claim 1, wherein said microfluidic substrate consists essentially of silicon.
`
`11. The device of Claim 1, further comprising a detector for measuring a physicochemical property of said
`
`25
`
`biological sample.
`
`12. The device of Claim 1, wherein said thermal transfer member comprises a metal bar in fluid
`
`communication with a plurality of water sources containing water at said at least two temperatures, said metal bar
`
`being in thermal communication with said at least a portion of said sample pathway.
`
`13. A method for conducting a biochemical or chemical process comprising:
`
`30
`
`cycling at least a portion of at least one sample flow pathway between at least two temperatures
`
`while a sample comprising the reagents for said biochemical or chemical process is flowing through said at
`
`least a portion of said at least one sample flow pathway.
`
`14. The method of Claim 13, wherein said sample flow pathway is located on a microfluidic substrate.
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`15. The method of Claim 14, wherein said sample flow pathway is in thermal communication with at least
`
`one thermal transfer member which cycles between said at least two temperatures while said sample is continuously
`
`flowing through said at least a portion of said at least one sample flow pathway.
`
`16. The method of Claim 15, wherein said thermal transfer member cycles through said at least two
`
`temperatures a plurality of times while said sample is continuously flowing through said at least a portion of said at
`
`least one sample flow pathway.
`
`17. The method of Claim 16, wherein said thermal transfer member cycles through said at least two
`
`temperatures from about 2 to about 35 times while said sample is continuously flowing through said at least a portion
`
`of said at least one sample flow pathway.
`
`18. The method of Claim 16, wherein at a portion of a plurality of sample flow pathways are
`
`simultaneously cycled between said at least two temperatures while a plurality of samples are simultaneously flowing
`
`through said sample flow pathways.
`
`19. The method of Claim 18, wherein said biochemical or chemical reaction comprises a nucleic acid
`
`amplification procedure.
`
`20. The method of Claim 19, wherein said nucleic acid amplification procedure comprises polymerase chain
`
`reaction.
`
`21. The method of Claim 19 further comprising determining the identity of at least one polymorphic
`
`nucleotide in the product of said nucleic acid amplification procedure.
`
`22. A process for carrying out biochemical protocols on at least one sample, comprising:
`
`feeding at least one channel with a continuous flow of a solution containing at least one sample;
`
`injecting at least one reagent from a reagent reservoir into said channel, thereby mixing said
`
`sample and said reagent: and
`
`transferring heat between at least one thermal support and at least one temperature regulated
`
`portion of said at least one channel.
`
`23. The process according to Claim 22, wherein said feeding comprises applying a pressure difference
`
`between a feed basin of said at least one channel and an outlet basin of said at least one channel.
`
`24. The process according to Claim 22, further comprising detecting at least one physicochemical
`
`parameter of said sample in said at least one channel.
`
`25. The process according to Claim 22, wherein a temperature of said solution is adjusted to a
`
`predetermined level when said solution runs through said at least one temperature regulated portion of said at least
`
`one channel.
`
`26. The process according to Claim 22, further comprising cycling said at least one thermal support through
`
`at least two different temperatures.
`
`27. The process according to Claim 26, wherein said cycling is repeated 1 to 35 times while solution is
`
`running through said at least one portion of said at least one channel.
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`28. The process according to Claim 22, wherein a plurality of samples separated by separators are
`
`sequentially introduced into said at least one channel.
`
`29. The process according to Claim 22, wherein said feeding, said injecting, and said transferring are
`
`carried out simultaneously on a plurality of channels arranged in parallel.
`
`30. A process for carrying out in continuous flow at least one temperature cycle on a solution containing at
`
`least one sample, comprising:
`
`feeding at least one channel with a continuous flow of said solution;
`
`running said solution through at least one temperature regulated zone; and
`
`cycling said at least one temperature regulated zone successively through a temperature cycle of
`
`10
`
`at least two temperatures in a predetermined temporal series, such that the solution undergoes said
`
`temperature cycle at least once in running through the at least one temperature regulated zone once.
`
`31. The process according to Claim 30, further comprising detecting at least one physicochemical
`
`parameter of said sample in said channel.
`
`32. The process according to Claim 30, wherein said feeding comprises applying a pressure difference
`
`15
`
`between a feed basin of said at least one channel and an outlet basin of said at least one channel.
`
`33. The process according to Claim 30, wherein said feeding is sequentially repeated with a plurality of
`
`samples separated by separators.
`
`34. The process according to Claim 30, wherein said feeding, said running and said cycling are carried out
`
`simultaneously on a plurality of channels arranged in parallel.
`
`20
`
`35. A process for amplifying nucleic acids, comprising:
`
`a)
`
`mixing at least one sample comprising said nucleic acids with reagents which are suitable
`
`for amplifying nucleic acids to form at least one reaction mixture:
`
`b)
`
`cl
`
`feeding at least one channel with a continuous flow of said at least one reaction mixture;
`
`running said at least one reaction mixture through at least one temperature regulated
`
`25
`
`zone; and
`
`d) cycling said temperature regulated zone through a temperature cycle of at least two
`
`temperatures in a predetermined temporal series, wherein the at least two temperatures, a duration of the
`
`temperature cycle, and a rate of said running are preselected such that said at least one nucleic acid sample
`
`undergoes a denaturation~hybridization-elongation cycle one or more times while flowing through said at
`
`30
`
`least one temperature regulated zone.
`
`36. The process according to Claim 35, wherein said feeding comprises applying a pressure difference
`
`between a feed basin of said at least one channel and an outlet basin of said at least one channel.
`
`37. The process according to Claim 35, wherein said channel is formed in a microfluidic substrate.
`
`38. The process according to Claim 35, wherein said microfluidic substrate consists essentially of silicon.
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`39. The process according to Claim 35, in which said feeding is sequentially repeated with a plurality of
`
`nucleic acid samples separated by separators.
`
`4D. The process according to Claim 35, in which steps a), b), c) and d) are carried out simultaneously on a
`
`plurality of channels arranged in parallel.
`
`41. A process for identifying in continuous flow at least one nucleotide in at least one target nucleic acid,
`
`comprising:
`
`a)
`
`feeding a channel with a continuous flow of a solution comprising said at least one target
`
`nucleic acid;
`
`b)
`
`injecting a microsequencing reagent comprising a microsequencing buffer, at least one
`
`microsequencing primer, at least one ddNTP and a polymerase into said channel, thereby mixing said nucleic
`
`acid solution and said reagent;
`
`c)
`
`running the solution through at least one temperature regulated zone in such a way as to
`
`produce at least one cycle comprising denaturation of said at least one target nucleic acid, hybridization of
`
`said nucleic acid with said at least one microsequencing primer, and incorporation of a ddNTP which is
`
`complementary to the nucleotide to be identified at a 3’ end of said primer; and
`
`d)
`
`identifying the nucleotide which has been incorporated at the 3' end of the
`
`microsequencing primer.
`
`42. The process according to Claim 41, wherein said feeding comprises applying a pressure difference
`
`between a feed basin of said channel and an outlet basin of said channel.
`
`43. The process according to Claim 41, further comprising amplifying said at least one target nucleic acid
`
`using the method of Claim 35 prior to performing said method for identifying at least one nucleotide.
`
`44. The process according to Claim 41, wherein the ddNTPs are labelled with fluorophores and wherein the
`
`fluorescence of the incorporated ddNTP is detected.
`
`45. The process according to Claim 44, in which said feeding, said injecting and said running are carried out
`
`simultaneously on a plurality of channels arranged in parallel.
`
`46. A process for detecting in continuous flow at least one nucleotide in at least one target nucleic acid,
`
`comprising:
`
`a)
`
`feeding a channel with a continuous flow of a solution containing at least one target
`
`nucleic acid;
`
`b)
`
`injecting the reagent for amplifying a region of the at least one target nucleic acid which
`
`carries at least one nucleotide to be detected into said channel from a first reagent reservoir;
`
`c)
`
`running the solution through at least one temperature regulated zone in such a way that
`
`the nucleic acid undergoes a denaturation-hybridization-elongation cycle one or more times;
`
`(1)
`
`injecting the reagent for purifying the amplification product into said channel from a
`
`second reagent reservoir;
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`e)
`
`running the solution through at least one temperature regulated zone to carry out a
`
`purification reaction:
`
`fl
`
`injecting the microsequencing reagent comprising the microsequencing buffer, at least
`
`one microsequencing primer, at least one ddNTP and a polymerase into said channel from a third reagent
`
`reservoir;
`
`g)
`
`running the reaction mixture through at least one temperature regulated zone in such a
`
`way as to produce at least one cycle comprising the denaturation of the target nucleic acid, the hybridization
`
`of said nucleic acid with the at least one microsequencing primer, and the incorporation of the ddNTP which
`
`is complementary to the nucleotide to be detected, at the 3’ end of said primer; and
`
`h)
`
`detecting at least one ddNTP which is incorporated at the 3' end of the microsequencing
`
`primer.
`
`47. The process according to Claim 46, wherein said feeding comprises applying a pressure difference
`
`between a feed basin of said channel and an outlet basin of said channel.
`
`48. The process according to Claim 46, wherein in steps cl and e), the temperature regulated zone is
`
`brought successively to at least two temperatures in a temporal series which forms at least one cycle.
`
`49. The process according to Claim 46, wherein the ddNTPs are labelled with fluorophores, and wherein in
`
`step b) the fluoresence of the incorporated ddNTP is detected.
`
`50. The process according to Claim 46, wherein the reagent for the purification comprises an exonuclease
`
`and an alkaline phosphatase.
`
`51. The process according to Claim 46, wherein steps a), b), c), d), e), fl, gland h) are carried out
`
`simultaneously on a plurality of channels arranged in parallel.
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`Figure 1
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`
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`Metal bar - PCR
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`902
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`
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`
`Figure 2
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