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Reaction vessel comprising electrically conducting polymer as a heating element

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Title: Reaction vessel comprising electrically conducting polymer as a heating element.
Abstract: A reaction vessel for conducting a chemical or biochemical reaction, such as a polymerase chain reaction wherein electrically conducting polymer is arranged to act as a heating element. The profile of the electrically conductive polymer differs in different regions of the vessel so as to control thermal gradients. The profile of the electrically conductive polymer may be arranged to either increase or reduce the thermal gradient. Reaction systems comprising combinations of vessels of the invention and apparatus for heating them, as well as particular reactions vessels are also described and claimed. ...


USPTO Applicaton #: #20110065150 - Class: 435 912 (USPTO) - 03/17/11 - Class 435 
Chemistry: Molecular Biology And Microbiology > Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition >Preparing Compound Containing Saccharide Radical >N-glycoside >Nucleotide >Polynucleotide (e.g., Nucleic Acid, Oligonucleotide, Etc.)

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The Patent Description & Claims data below is from USPTO Patent Application 20110065150, Reaction vessel comprising electrically conducting polymer as a heating element.

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US 20110065149 A1 20110317 1 38 1 20 PRT artificial sequence artificial linker which is completely synthesized 1 Gly Met Gly Leu Ser Trp Ala Gly Gln Leu Pro Arg Phe Pro Gln Arg 1 5 10 15 Ala Ser Gln Gly 20 2 60 DNA artificial sequence artificial linker which is completely synthesized 2 ggtatgggtt tgtcttgggc tgggcagctg cctaggtttc ctcagcgtgc tagtcaaggg 60 3 20 PRT artificial sequence artificial linker which is completely synthesized 3 Lys Gly Arg Gln Gln Leu Gln Leu Cys Ala Pro Phe Asp Trp Cys Met 1 5 10 15 Phe Asp Ala Asn 20 4 60 DNA artificial sequence artificial linker which is completely synthesized 4 aagggtcggc aacagctgca gttgtgtgcg ccttttgatt ggtgtatgtt tgatgctaat 60 5 20 PRT artificial sequence artificial linker which is completely synthesized 5 Trp Val Trp Ser Ser Arg Gly Gln Arg Ser Phe Arg Pro Ser Gly Arg 1 5 10 15 Thr Val Pro Leu 20 6 60 DNA artificial sequence artificial linker which is completely synthesized 6 tgggtttgga gttcgcgggg gcagaggtct tttcggcctt cggggcggac ggtgccgctt 60 7 20 PRT artificial sequence artificial linker which is completely synthesized 7 Lys Val Val Leu Trp Thr Thr Arg Val Arg Asp Arg Gly His Thr Ser 1 5 10 15 Thr Met Trp Ser 20 8 60 DNA artificial sequence artificial linker which is completely synthesized 8 aaggttgttc tttggactac gcgtgttagg gataggggtc atacgtcgac gatgtggagt 60 9 20 PRT artificial sequence artificial linker which is completely synthesized 9 Ala Asp Gly His Cys His Leu Lys Asn Phe Pro Leu Lys Pro Pro Pro 1 5 10 15 Tyr Phe Ser Val 20 10 60 DNA artificial sequence artificial linker which is completely synthesized 10 gcggatgggc attgtcatct gaagaatttt cctttgaagc ctccgcctta tttttcggtt 60 11 16 PRT artificial sequence artificial linker which is completely synthesized 11 Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys 1 5 10 15 12 48 DNA artificial sequence artificial linker which is completely synthesized 12 ctactaaaaa aactactaaa aaaactacta aaaaaactac taaaaaaa 48 13 15 PRT artificial sequence artificial linker which is completely synthesized 13 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 14 45 DNA artificial sequence artificial linker which is completely synthesized 14 ggtggaggcg gttccggcgg aggtggctcc ggcggtggcg gatcc 45 15 119 PRT Mus musculus 15 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Val Leu Gly Arg Gly Tyr Gly Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 16 357 DNA Mus musculus 16 gatgtacagc ttcaggagtc aggacctggc ctcgtgaaac cttctcagtc tctgtctctc 60 acctgctctg tcactggcta ctccatcacc agtggttatt actggaactg gatccggcag 120 tttccaggaa acaaactgga atggatgggc tatataaggt acgacggtag caataactac 180 aacccatctc tcaaaaatcg aatctccatc actcgtgaca catctaagaa ccagtttttc 240 ctgaaattga attctgtgac tcctgaggac acagctacat attactgtgc aagagtattg 300 ggacggggct atggtttgga ctactggggt caaggaacct cagtcaccgt ctcctca 357 17 111 PRT Mus musculus 17 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Thr Tyr Ser Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro 35 40 45 Lys Leu Ile Lys Tyr Val Ser Asn Leu Glu Ser Gly Val Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro 65 70 75 80 Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp Glu 85 90 95 Ile Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 18 333 DNA Mus musculus 18 gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60 atctcatgca gggccagcca aagtgtcagt acatctacct atagttattt acactggtac 120 caacagagac caggacagcc acccaaactc atcaagtatg tatccaacct agaatctggg 180 gtccctgcca ggttcagtgg cagtgggtct gggacagact tcaccctcaa catccatcct 240 gtggaggagg aggatactgc aacatattac tgtcagcaca gttgggagat tcctccgacg 300 ttcggtggag gcaccaagct ggaaatcaaa cgg 333 19 94 DNA artificial sequence artificial linker which is completely synthesized 19 cagtcaccgt ctcctcannk nnknnknnkn nknnknnknn knnknnknnk nnknnknnkn 60 nknnknnknn knnknnkgac attgtgctga caca 94 20 41 DNA artificial sequence artificial linker which is completely synthesized 20 ctcccatggc cgatgtacag cttcaggagt caggacctgc c 41 21 37 DNA artificial sequence artificial linker which is completely synthesized 21 atatatgcgg ccgcccgttt gatttccagc ttggtgc 37 22 30 DNA artificial sequence artificial linker which is completely synthesized 22 tgaggagacg gtgactgagg ttccttgacc 30 23 30 DNA artificial sequence artificial linker which is completely synthesized 23 gacattgtgc tgacacagtc tcctgcttcc 30 24 45 DNA artificial sequence artificial linker which is completely synthesized 24 ggtggaggcg gttccggcgg aggtggctcc ggcggtggcg gatct 45 25 60 DNA artificial sequence artificial linker which is completely synthesized 25 tgggtttgga gttcgcgtgg gcagcgttct tttcgtcctt cggggcgtac ggtgccgctt 60 26 24 DNA artificial sequence PCR primer 26 caggtccagc tgcagcagtc tgga 24 27 27 DNA artificial sequence PCR primer 27 tgaggagact gtgagagtgg tgccttg 27 28 30 DNA artificial sequence PCR primer 28 gatgttgtga tgacccaaag tccactctcc 30 29 30 DNA artificial sequence PCR primer 29 ttttatttcc agcttggtcc cccctccgaa 30 30 79 DNA artificial sequence PCR primer 30 ctctcacagt ctcctcaggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 60 cagatgttgt gatgaccca 79 31 79 DNA artificial sequence PCR primer 31 ctctcacagt ctcctcaggt ggaggcggtt ccggcggagg tggctccggc ggtggcggat 60 ccgatgttgt gatgaccca 79 32 25 DNA artificial sequence PCR primer 32 tttccatggc ccaggtccag ctgca 25 33 29 DNA artificial sequence PCR primer 33 ttttttgcgg ccgcttttat ttccagctt 29 34 52 DNA artificial sequence PCR primer 34 tcatttatcg tcatcgtctt tgtagtcttt tatttccagc ttggtccccc ct 52 35 79 DNA artificial sequence PCR primer 35 ccaagctgga aataaaaggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 60 cacaggtcca gctgcagca 79 36 41 DNA artificial sequence PCR primer 36 ttttttgcgg ccgctcattt atcgtcatcg tctttgtagt c 41 37 79 DNA artificial sequence PCR primer 37 ccaagctgga aataaaaggt ggaggcggtt ccggcggagg tggctccggc ggtggcggat 60 cccaggtcca gctgcagca 79 38 48 DNA artificial sequence artificial linker which is completely synthesized 38 ctactaaaaa aactactaaa aaaactacta aaaaaactac taaaaaaa 48 US 20110065150 A1 20110317 US 12671783 20080801 12 GB 0715170.7 20070803 20060101 A
C
12 P 19 34 F I 20110317 US B H
20060101 A
C
12 M 1 00 L I 20110317 US B H
20060101 A
C
12 M 1 22 L I 20110317 US B H
20060101 A
C
12 M 1 24 L I 20110317 US B H
US 435 912 4352891 4353051 4353041 REACTION VESSEL COMPRISING ELECTRICALLY CONDUCTING POLYMER AS A HEATING ELEMENT Jones Ross Peter
Cambridge GB
omitted GB
Williamson Roger James
Hertfordshire GB
omitted GB
Gregory Richard George
Hertfordshire GB
omitted GB
Gutsell Graham
Wiltshire GB
omitted GB
Lee Martin Alan
Wiltshire GB
omitted GB
Squirrell David James
Wiltshire GB
omitted GB
ENIGMA DIAGNOSTICS LIMITED 03
Wiltshire GB
WO PCT/GB08/02627 00 20080801 20101130

A reaction vessel for conducting a chemical or biochemical reaction, such as a polymerase chain reaction wherein electrically conducting polymer is arranged to act as a heating element. The profile of the electrically conductive polymer differs in different regions of the vessel so as to control thermal gradients. The profile of the electrically conductive polymer may be arranged to either increase or reduce the thermal gradient. Reaction systems comprising combinations of vessels of the invention and apparatus for heating them, as well as particular reactions vessels are also described and claimed.

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The present invention relates to reaction vessels useful in chemical and biochemical reactions which are required to undergo controlled heating and/or cooling, in particular, vessels which are required to undergo thermal cycling, where a sequence of different temperatures are required.

A particular example of such a reaction are a number of nucleic acid amplification methods, in particular the polymerase chain reaction (PCR). As is well known, in this reaction, exponential amplification of nucleic acids is achieved by cycling the sample containing or suspected of containing the target nucleic acid through an iterative sequence of different temperatures in the presence of specifically designed primer sequences and polymerase enzymes able to extend those primer sequences. These temperatures represent the temperatures necessary for nucleic acid denaturation (and generally requires temperatures of about 95° C.), primer annealing (at a lower temperature for example at about 55° C.) and primer extension (which may require and intermediate temperature for example of about 74° C.).

There is frequently a need to obtain the results of a PCR reaction quickly, for example in cases of environmental contamination which may be the result of hostile activity. However, even in a clinical or diagnostic situation, the production of quick results can be helpful, in particular where patient compliance or return can be problematic.

Clearly, for fast PCR, the sample must be rapidly heated and cooled. This is facilitated by making the sample small to reduce its thermal mass and by minimising the distances over which heat must be transferred. The same considerations must be applied to the container of the sample.

Thus, a number of examples of apparatus designed to carry out PCR reactions utilize reaction vessels which comprise a capillary tube format (ie long and thin WO 2005/019836) or as a planar structure (flat and thin) (WO2006024879), the content of which are incorporated herein by reference.

A variety of heating systems are utilized in order to achieve rapid PCR. These include for example fluid based systems in which hot fluid such as air is fed to the container of the sample for heating purposes, and non-heated fluid is supplied to effect cooling (see for example U.S. Pat. No. 6,787,338 and WO2007/054747, the content of which is incorporated herein by reference).

In an alternative type of apparatus, electrically conducting polymer (ECP) is used as both the heating element and in some cases also the container (see WO 98/24548, the content of which is also incorporated herein by reference).

The ECP acts as a resistive heater and so it is required to be connected to an electrical supply by way of electrical connections. As an inevitable consequence of reducing the thermal mass of the sample and facilitating heat transfer into and out of it, the means of connecting and locating the ECP can have significant thermal effects upon it.

A particular problem is the formation of temperature gradients as heat can be conducted both out from and in to the extremities of the ECP tube, through the electrical connections, as it is heated and cooled, respectively.

This problem has been addressed in some instances by examining the electrical connections themselves and in particular, the mountings for the electrodes. These must be electrically insulating and are preferably also thermally insulating. However, the property of thermal insulation is in itself insufficient.

Electrical connectors (or electrodes) that are thermally insulating heat and cool slowly which has the effect of making them important contributors to the formation of longitudinal temperature gradients during rapid thermal cycling. The mountings must also therefore have a low thermal mass as well as being thermal insulators. This may be achieved by placing insulating materials that have been structured to reduce their thermal mass whilst retaining the physical integrity needed to support the electrodes. Such structuring, in its essence, requires the inclusion of air gaps in the mountings. This may be achieved by using foam materials, such a honeycomb or reticulated foam to form a mount for the electrode. The mounts are suitable in the form of a pillared or corrugated mount for the electrical connector (as described for example in WO2005/0011834, WO2004/045772 and copending British Patent Application No. 0623910.7).

In PCR, it would be ideal to have all parts of the sample at the same controlled temperature all of the time. This is extremely difficult in a system that is being rapidly heated and cooled. In the capillary format, both radial and longitudinal temperature gradients are formed.

The applicants have found however that the profile of the ECP can be adjusted to control the thermal gradient

A first aspect of the present invention provides a reaction vessel heating system, which comprises electrically conducting polymer, arranged to act as a heating element for a reaction vessel, wherein the profile of the electrically conductive polymer differs in different regions of the vessel so as to control thermal gradients therealong.

The ECP profile may be arranged to have the effect of reducing thermal gradients. This is particularly suitable for reaction vessels used for thermal cycling reactions, for example PCR.

In one embodiment, the profile of the ECP is itself adjusted to vary its radial thickness so that the resistance heating is distributed unevenly in a way so as to reduce gradients. This may be done empirically, in relation to any particular vessel type, by determining the gradient profile which occurs and adjusting the thickness of the ECP in various regions of the vessel accordingly. Typically, the profile will be tapered, being narrower at the bottom of the vessel than the top. Instead of varying the radial thickness, the area of contact between the vessel and ECP may be varied.

The thermal gradients may be further reduced by providing the vessel with at least one wall which comprises a highly thermally conducting layer. The highly thermally conducting layer may comprise a metallic layer, for example aluminium. The electrically conductive polymer is insulated from the metallic layer by means of an insulating layer therebetween, for example a layer of anodised aluminium, or is a polymer layer, such as parylene or a derivative thereof. Preferably the vessel further comprises an inner non metallic layer to improve biocompatability.

The reaction vessel may be an elongate vessel. In a preferred embodiment, the reaction vessel comprises a tube which is sealed at one end, wherein the end is of a transparent material. The reaction vessel may comprise a capillary vessel or a flattened capillary vessel.

In another embodiment, the ECP profile is arranged to have the effect of increasing thermal gradients. A thermal gradient is thus created along the vessel.

A vessel with a thermal gradient may be used for the culture of biological materials, with different regions along the thermal gradient being optimal for different biological material. The vessel may comprise a petri dish, cuvette, chemostat, shake flask, universal container, bijou.

The profile may be adapted by changing the radial thickness of ECP. Alternatively, the area of contact between the ECP and the vessel may be varied.

A second aspect of the present invention provides a method for carrying out a chemical or biochemical reaction which requires at least one heating step, said method comprising placing chemical or biochemical reagents into a reaction vessel according to the present invention and heating said reagents so as to bring about a chemical or biochemical reaction.

Preferably the reaction requires thermal cycling. More preferably the reaction is a polymerase chain reaction.

A third aspect of the present invention provides a method for mixing reagents in a vessel, said method comprising placing said chemical or biochemical reagents in a reaction vessel according to the present invention and heating the heating the vessel so as to bring about a temperature gradient to thereby create convection.

Such modifications may be included in vessels which include or utilise ECP resistance heaters, irrespective of the presence or otherwise of the highly thermally conducting layer, and such vessels form yet a further aspect of the invention.

According to a fourth of the present invention there is provided a reaction vessel for conducting a chemical or biochemical reaction, wherein at least one wall of said vessel comprises a layer of a highly thermally conducting material, (in particular a metallic layer) and an inner non-metallic layer.

For the avoidance of doubt, the term “layer” as used herein refers to any essentially laminar arrangement of material, including both self-supporting layers as well as coatings. Layers or coatings which are not self supporting, will generally conform to the shape of the relevant substrate, and so for example may in practice may be any shape, including in particular tubular. Similarly, self-supporting layers may take whatever form is particularly convenient in relation to the context in which they are used.

Reaction vessels of the invention have good thermal conductivity as a result of the presence of the highly thermally conducting layer of the wall, and therefore can be used in reactions where temperature control, or in particular temperature cycling with good temperature uniformity is important. Therefore, they may be particularly useful in reactions such as nucleic acid amplification reactions which involve thermal cycling such as the polymerase chain reaction (PCR). The good thermal conductivity means that significant temperature gradients through the vessel do not form, or are rapidly evened out if they do occur, so that the temperature profile along and across the sample is made flatter (more homogeneous).

Generally, metal walled vessels have not be used hitherto in reaction vessels because they are generally chemically reactive in particular with biological molecules such as nucleic acids and proteins, and so the metal interferes with reagents in the vessel and so disrupts the reaction. However, the applicants have found that this problem can be overcome by the provision of an inner non-metallic layer, in contact with the thermally conducting layer such as the metallic layer so as to effectively form a composite.

The vessel is suitably an elongate vessel, with the layered wall forming at least one of the long walls so as to increase the surface area of the metal containing wall which is in the proximity of a reagent in the vessel. In particular, the vessel is a capillary vessel or a flattened capillary vessel, where the length is selected to accommodate the volume of the sample and inner diameters are small. In particular, the inner diameter of a capillary tube is in the range of from 0.2 to 2 mm. The thickness of the wall is generally from about 0.1 to about 1.5 mm.

Examples of such vessels are described for example in WO2004/054715, U.S. Pat. No. 6,015,534 and WO 2005/019836, the content of which is incorporated herein by reference.

Such vessels effectively comprise a single radial side wall and this suitably comprises a metallic layer over substantially all, and preferably all its area.

Where flattened tubes are used, they may be of a shape described in WO2006024879, the content of which is incorporated herein by reference. Specifically, such vessels have a width:depth ratio of about 2:1 or more, for example, of 3:1 or more. Typically the width of the vessels may be of the order of 1 mm or less for example 0.8 mm or less, whereas the depth is generally 0.5 mm or less, and suitably less than 0.3 mm. The vessels may be tapered.

In these cases, at least one side wall comprises a metallic layer, and preferably all side walls comprise a metallic layer. The lower wall may also have this construction, although in many instances, it is preferred that the lower wall, which forms the base of the vessel is of a transparent material such as glass or a transparent polymer so that the contents of the reaction vessel can be optically monitored during the reaction. This is particularly helpful in the case of the use of the so-called “real-time” PCR reactions where optical signals, in particular fluorescent signals from signalling reagents added to the PCR reaction, produce a variable signal as the reaction progresses, so that the progress of the reaction can be monitored. Such monitoring gives rise to the option of quantifying the amount of target nucleic acid within the initial sample, so providing further information which may be of use, for example in diagnostics, in determining the seriousness of a particular condition.

Suitable non-metallic materials for the inner non-metallic layers may include polymeric materials or glass or even a passivated layer created through anodisation of a metal, or similar process, or combinations of these. In particular, however, the inner non-metallic layer is a polymer or glass or combination of these.

In a particular embodiment, the inner non-metallic layer is a glass layer, since glass is generally well recognised as being compatible with many biochemical and chemical reactions including the polymerase chain reaction.

However, polymeric materials such as polyurethane, polyethylene, polypropylene, or polycarbonates, as well as silicones which are compatible with the sample and with the particular reaction being carried out within the reaction vessel.

Such inner layers will generally be rigid and supporting structures, which may be formed by processes such as injection or extrusion moulding and the like. These may then be coated with a metallic layer, or they may be extruded or formed directly onto the metallic layer.

However, if necessary or required a thin layer for example of polymeric material may deposited on the metallic layer for example using techniques such as vapour deposition, liquid phase deposition or plasma polymerisation to provide a relatively thin layer which may itself constitute the inner non-metallic layer. Alternatively, such a thin layer may be applied to a different inner non-metallic layer as described above to form a composite structure.

A particularly suitable polymeric layer of this type is formed of parylene or derivatives thereof. Parylene is a generic name applied to polyxylylene as for example as described in U.S. Pat. No. 3,343,754, the content of which is incorporated herein by reference.

Compounds of this type can be represented by the general formula (I)

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where is R is a substituent group, m is 0 or an integer of from 1 to 3 and n is sufficient for the compound to be a polymer.

Where m is greater than 1, each R group may be the same or different.

In one embodiment, m is 0.

Suitable substituent groups R include but are not limited to R1, OR1, SR1, OC(O)R1, C(O)OR1, hydroxyl, halogen, nitro, nitrile, amine, carboxy or mercapto and where R1 is any hydrocarbon group and where R1 may be optionally substituted by one or more groups selected from hydroxyl, halogen, nitro, nitrile, amine or mercapto.

Suitable hydrocarbon groups include alkyl groups such as straight or branched chain C1-10alkyl groups, alkenyl groups such as straight or branched C2-10alkenyl groups, alkynyl groups such as straight or branched C2-10alkynyl groups, aryl groups such as phenyl or napthyl, aralkyl groups such as aryl (C1-10) alkyl for instance benzyl, C3-10cycloalkyl, C3-10cycloalkyl (C1-10) alkyl, wherein any aryl or cycloalkyl groups may be optionally substituted with other hydrocarbon groups and in particular alkyl, alkenyl or alkynyl groups as described above.

Particular examples of groups R include alkyls such as methyl, ethyl, propyl, butyl or hexyl, which may be optionally substituted with hydroxy, halo or nitrile such as hydroxymethyl or hydroxyethyl, alkenyls such as vinyl, aryls in particular phenyl or napthyl which may be optionally substituted by halo or alkyl groups such as halophenyl or C1-4alkylphenyl, alkoxy groups such as methoxy, ethoxy, propoxy, carboxy, carbomethoxy, carboethoxy, acetyl, propionyl or butyryl.

In particular, R is selected from halogen (particularly chlorine or bromine), methyl, trifluoromethyl ethyl, propyl, butyl, hexyl, phenyl, C1-4alkylphenyl, naphthyl, cyclohexyl and benzyl.

Examples of such polymers are sold as “Parylene”. Particular variety of parylene which may be obtained include Parylene N (where m is 0), Parylene C (where m is 1 and R is chloro), Parylene F (where m is 1 and R is trifluoromethyl) and Parylene D (where m is 2 and each R is chloro).

Parylene is a particularly convenient polymeric material for providing an internal coating for the metallic surface, as it may be readily applied using a vapour deposition process. In this process a solid dimer of formula (II)

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where R and m are as defined above, is placed into a suitable vaporisation chamber in solid form. When the chamber and heated under reduced pressure, for example to temperatures of about 150° C. at low pressure, for example of about 1 mmHg, the diner vapourises. The vapour is then transferred into a pyrolysis chamber where the temperature is much higher, for example at about 650° C. and the pressure is for example of 0.5 mmHg, Pyrolysis occurs so as to cause the formation of a reactive monomeric species of formula (III).

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If this is allowed to pass into a further chamber containing the item to be coated which is at ambient temperature, but also at low pressure, for example of 0.1 mmHg, polymerisation of the species (III) occurs on the surface of the object, so that a coating of the polymer of formula (I) above is produced.

The species (III) condenses on the surface in a polycrystalline fashion, providing a coating that is conformal and pinhole free. This is important to ensure that any sample within the reaction vessel is isolated from the metallic layer.

Compared to liquid processes, the effects of gravity and surface tension are negligible—so there is no bridging, thin-out, pinholes, puddling, run-off or sagging. And, since the process takes place at room temperature, there is no thermal or mechanical stress on the object.

Parylene is physically stable and chemically inert within its usable temperature range, which includes the temperatures at which PCR reactions are conducted. Parylene also provides excellent protection from moisture, corrosive vapours, solvents, airborne contaminants and other hostile environments.

It is widely used in the electronics industry to coat and protect electronic components. However, the applicants are the first to find that parylene is compatible with chemical or biochemical reactions and in particular with the PCR reaction, and the use of parylene for coating reaction vessels and in particular PCR reaction vessels is described and claimed in the applicants copending patent application of even date.

In the vessel of the apparatus, the metallic layer effectively forms a thermal shunt, conducting heat rapidly from one part of the reaction vessel to another. Thus it minimises the build-up of thermal gradients in the vessel and therefore in the sample during the reaction, which is important in ensuring that the reaction proceeds efficiently and well.

Thus vessels comprising a highly thermally conducting material most suitably comprise a material which has a thermal conductivity in excess 15 W/mK. Materials having this property will generally be metallic in nature, but certain polymers, in particular those known as “cool polymers” or polymers containing thermally conducting ceramics such as boron nitride as well as diamond, may have the desired level of thermal conductivity. In particular however, the highly thermally conducting material is metallic, which may be of any suitable metal or metal alloy including aluminium, iron, steel such as stainless steel, copper, lead, tin or silver. In particular, the metallic layer comprises aluminium.

The reaction vessel in accordance with the invention, may be heated by any suitable heating means, and as a result of the presence good thermal conductivity of the walls of the vessel due to the presence of the highly thermally conducting layer, the heat will be readily transferred to the vessel contents.

Thus the vessels are suitable for use in a wide range of apparatus in particular thermal cycling equipment. These may be heatable and/or coolable using a number of different technologies, including the use of fluid heaters and coolers such as air heaters and coolers in particular those heated by halogen bulbs, as described for example in U.S. Pat. No. 6,787,338 and WO2007/054747, the content of which is incorporated by reference, as well as in vessels using ECP as resistive heating elements, for example as described in WO 98/24548 and WO 2005/019836 as will be discussed further below. The vessels may also be used in more conventional devices such as solid block heaters that are heated by electrical elements. For cooling the apparatus may incorporate thermoelectric devices, compressor refrigerator technologies, forced air or cooling fluids as necessary. However, where the vessels of the invention comprise a metallic layer, this means that they may also be capable of being heated using for example induction methods. Apparatus used to heat vessels of the invention in this way will have the facility to heat the vessel by electromagnetic induction, for example by using a high-frequency alternating current (AC) to induce eddy currents within the metal. Resistance of the metal to these currents leads to Joule heating of the metal. Heat is also generated by magnetic hysteresis losses. For use in induction heating apparatus, it will be clear that the metallic layer within the vessel should be of a suitable material to allow it to be heated in this way, and so for example iron metallic layers may be preferred to say stainless steel or copper.

Reaction systems comprising combinations of reaction vessels as described above, and apparatus which is able to accommodate said reaction vessel, and which comprises a heating system adapted to controllably heat and cool said vessel, in particular using any of the methods discussed above, form a further aspect of the invention.

When the reaction vessels of the invention are utilised in combination with resistive heating elements, such as ECP, it is necessary to ensure that where the highly thermally conducting layer is also electrically conducting, such as a metallic layer, this is electrically insulated from the resistive heater in order to prevent short circuits etc. The applicants have found that it is possible to passivate the surface of a metal layer so that it is electrically isolated from the ECP, but still in good thermal contact. For example in the case of an aluminium metallic layer, anodisation of any surface of the aluminium layer which is to be in contact with the resistive heater such as the ECP provides such insulation.

Alternatively, a parylene layer, preparable as described above may be applied to the surface of the highly thermally conducting layer such as the metallic layer which contacts the ECP so as to provide an effective electrically insulating layer. Such layers have the benefit that they do not significantly add to the size or thermal mass of the vessel.

The ECP is suitably arranged as a sheath or coating arranged outside the highly thermally conducting layer and the electrically insulating layer thereof, as described in WO 98/24548. By keeping the elements of the reaction vessel small, in particular as thin layers, the thermal mass of the vessel remains low, and so fast heating and cooling can take place as is required for rapid PCR.

Thus in a particularly preferred embodiment, a wall of the reaction vessel, and suitably the entire side walls of the vessel comprise an inner non-metallic layer, for example of glass or a polymeric material. This is covered by a metallic layer as described above, which is itself covered by an electrically insulating layer, for example a layer of anodised aluminium or parylene or a derivative thereof as described above. Outside of this layer is suitably provided a layer of electrically conducting polymer. Such vessels are generally intended to be disposable after a single use.

The electrically conducting polymer needs to be connected to an electrical supply, and so electrical connections, which may be integral with the vessel, are suitably provided at each end of the ECP.

The ECP elements used in the vessels as resistive heating elements can be manufactured by various processes, but most a convenient process involves injection moulding of the polymer. However, in the process of injection moulding, the material tends to form an outer polymer-rich skin that may creates at least a partial electrically insulating barrier to any external means of making electrical contact.

In such cases, the applicants have found that it is helpful to break through the insulating skin and make electrical contact with the bulk material in order to increase the efficiency of the heating system.

Thus, in a particular embodiment, the reaction vessel as described above, is connectable to an electricity supply by means of barbed electrical contacts which pierce the surface of the electrically conducting polymer. These are suitably integral with the vessel.

Such barbed connectors may take various forms depending upon the particular configuration of the reaction vessel itself. In particular, where the vessel is of a generally tubular configuration, suitable barbed connectors may take the form of annular metal rings with inwardly projecting barbs or the like, similar in design to “Starlock Washers”.

The inwardly projecting barbs will cut through the insulating skin to make electrical contact with the bulk conducting material, as well as hold the ring in position. The outer portion will present a metallic surface for electrical interconnection, and so apparatus intended to accommodate the vessels will be configured appropriately.

Furthermore, the barbs provide effectively a scalloped edge which helps to reduce the size and therefore the thermal mass of the connectors and also, reduces the contact area with the ECP. This has the further advantage of further minimising heat exchange between the electrical connectors and the ECP, so further assisting in reducing unwanted thermal gradient formation.

The connectors and particularly the barbs thereof, are suitably constructed of a material which have high mechanical strength, so that the barbs can be sharpened to enhance the penetration ability. Whilst many metals are able to fulfil this function, a particularly suitable material for the connectors has been found to be stainless steel. This not only has the required mechanical strength and electrical conductivity, but also, it has a high corrosion resistance, at 16 W/mK, a surprisingly low thermal conductivity compared to many other metals (cf Copper @ 399 W/mK, Aluminium @ 237 W/mK). By using this material, and by designing the connectors so that their area is as small as possible, helps to reduce unwanted thermal effects at the electrodes.

Connections of this type are cost effective to manufacture, which will be a particular advantage in cases where the proposed PCR vessel is to be a high volume disposable item.

They may, in fact, be used in connection with any reaction vessel or device which includes ECP as a resistive heater, and such reaction vessels and devices form a further aspect of the invention.

As discussed briefly above, the apparatus into which the vessel is accommodated for use suitably is provided with mounts of a material which is both an electrical and thermal insulator (i.e the material absorbs and releases heat slowly) and also have a low thermal mass. This can be achieved by making the electrode mounts “air-like” ie formed as foam, skeletal or honeycomb structures. However they must be able to physically support and position the electrodes, and therefore they must have a degree of rigidity, firmness and/or resilience.

Thus in a particular embodiment of the apparatus for thermally cycling the contents of the reaction vessel as described above comprises a solid foam material arranged in direct contact with at least one of said electrical contacts.

The solid foam material acts as an insulator, preventing heat flows and particularly heat loss through the electrical contacts. As a result, a more uniform temperature can be maintained within the reaction vessel. By utilising specifically a solid foam in preference to a conventional solid insulator, such as a solid plastics insulator, the performance of the apparatus is significantly enhanced. Thermal gradients set up within the reaction vessel can be further reduced.

It is believed that this is due to the fact that foam-like materials have a lower thermal mass than solids. Therefore, in addition to providing insulation preventing the flow of heat into and out of the reaction vessel through the electrical contact, they do not significantly hinder the heating and cooling process.

In contrast, solid insulators with significant thermal mass were found to heat up and cool down slowly (rather than not at all), thereby acting as sources or sinks of heat depending on their temperature relative to the sample. This was found to be disadvantageous in this context.

Suitable solid foam materials include metal, glass, carbon, polymer, ceramic foams or composites made of several of these.

Particular examples of such foam materials are polymeric foams such as polyurethane or polystyrene foams.

In a particular embodiment, the foam material is a ceramic foam. Ceramic foams generally comprise inorganic, non-metallic materials (such as metal oxides, silicides, nitrides, carbide or borides) with a crystalline structure, which have usually been processed at a high temperature at some time during their manufacture.

Many such foams are now commercially available. They have been developed mainly for the aerospace industry where their utility as insulators is a result of their light weight.

Solid foams generally comprise solids which have many gas bubbles trapped within them. They may be rigid or pliable in nature, but are preferably rigid so as to support the electrical contacts. Where they are pliable, the foam material suitably has a good “shape memory”.

Various forms and types of solid foam materials are known. Some are known as “refractory” foams. They are made by various methods depending upon the nature of the material used. For instance, solid foams comprising polymers may be readily prepared including foaming agents into the preparation process, as is well understood in the art. Syntactic foams and self-foamed materials such as foam glass may be prepared in a similar way.

Other solid foams may utilise a foamed polymer as the basic starting material and materials are essentially coated onto these or onto carbonaceous skeletons formed by pyrolysis of the polymer foam. For example, ceramic foams can be produced by coating a polymer foam or a carbon skeleton derived from it, with an appropriate binder and ceramic phases, and then sintering at elevated temperatures. Metallic foams may be formed by electrolytically depositing the metal onto a polymer foam, utilizes an electrodeless process for the deposition of a metal onto the polymer foam precursor via electrolytic deposition.

Suitably the solid foam material is of a material, which has no fluorescent or phosphorescent properties, even when illuminated with a light source. This means that it may not interfere with the fluorescent signalling or labelling systems that are frequently utilised for detecting the products of an amplification reaction. Such systems may be used to detect the product of amplification either at the end point of the reaction, or, increasingly, in “real-time” as the reaction progresses. These systems, which include the well known “Taqman™” system as well as other systems such as those described for example in Homogeneous fluorescent chemistries for real-time PCR. Lee, M. A., Squirrell, D. J., Leslie, D. L. and Brown, T. in Real-time PCR: an essential guide, J. Logan, K. Edwards & N. Saunders eds., Horizon Scientific Press, Wymondham, p. 31-70, 2004, the content of which is incorporated herein by reference. For instance, generic methods utilise DNA intercalating dyes that exhibit increased fluorescence when bound to double stranded DNA species. Fluorescence increase due to a rise in the bulk concentration of DNA during amplifications can be used to measure reaction progress and to determine the target molecule copy number. Furthermore, by monitoring fluorescence with a controlled change of temperature, DNA melting curves can be generated, for example, at the end of PCR thermal cycling.

However, when the material has a degree of fluorescence, this may be obviated by dying, coating or inking the foam before use.

The solid foam material is arranged in contact with at least one and preferably both of the electrical contacts. Suitably sufficient foam material is arranged so that in use, it effectively isolates the electrical contacts from environmental effects. This will generally be achieved by arranging the solid foam material in contact with at least the remote edges of the electrical contacts.

If desired, a solid insulator material may also be provided and arranged to contact the solid foam material.

Suitable solid insulator materials are well known in the art, and include polymeric or fibrous materials. In particular the solid insulator is a polymeric insulator such as an acetal homopolymer resin such as Delrin™, acrylonitrile-butadiene-styrene terpolymer (ABS) or polytertrafluoroethylene (PTFE).

The solid insulator may be arranged to contact a substantial portion, for example at least one side of the solid foam material. This additional insulation will protect the solid foam material itself from changes in environmental temperature and so enhance the overall reliability of the system. Suitably the additional solid insulator is provided so as to effectively isolate the reaction vessel from the external environment when in use. The precise arrangement of the solid insulator therefore will vary depending upon the nature of the reaction vessel and the manner in which it is used.

As mentioned above, example of mounts which include foam-like materials are described for example in WO2005/0011834, WO2004/045772, where the foam materials have a degree of resilience or compliance to allow them to hold the connectors firmly, or co-pending British Patent Application No. 0623910.7, where firm foam-like materials may be used, which are sprung-loaded to ensure that a sufficiently firm hold on the connectors is achieved.

Reaction vessels as described above and reaction systems comprising them can be used in chemical and biochemical reactions as required. Thus, in a further aspect, the invention provides a method for carrying out a chemical or biochemical reaction which requires at least one heating step, said method comprising placing chemical or biochemical reagents into a reaction vessel as described above, and heating said reagents so as to bring about said chemical or biochemical reaction. In particular, the vessel is positioned into apparatus specifically designed to hold it, and to heat and/or cool it as required. In particular, the apparatus comprises a thermal cycler as described above, and the reaction requires thermal cycling, in particular is a polymerase chain reaction.

The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings in which:

FIG. 1 shows a section through a reaction vessel according to the invention;

FIG. 2 shows an enlarged view of the portion of the reaction vessel shown in claim 1, which incorporates the elements of the invention;

FIG. 3 is a perspective end view of the reaction vessel of FIG. 1; and

FIG. 4 shows an electrical connection used in the embodiment of FIG. 3; and

FIG. 5 is a schematic diagram showing a vessel as shown in FIG. 1 in position in a thermal cycling apparatus.

FIG. 6 illustrates a sample vessel coated with ECP, showing tapering of the ECP layer;

FIG. 7 is a graph illustrating the internal temperatures of a sample vessel at a thermopile setting of 43° C.;

FIG. 8 is a graph illustrating the internal temperatures of a sample vessel at a thermopile setting of 63° C.;

FIG. 9 is a graph showing the effect of tapering the ECP at different thermopile settings; and

FIG. 10 is a graph showing the different temperatures recorded on a tapered section of ECP.

The reaction vessel shown in FIG. 1 is of the same general type as those described in for example WO2005/019836, which is intended for use in an apparatus for conducting a PCR reaction.

The vessel comprises a plastics body (1) with an upper sample receiving portion (2) with a relatively wide mouth so that reagents can, with ease, be added. In the illustrated embodiment, the upper portion includes projecting flanges (6) which are able to interact with a lifting arm in an apparatus such as that described in WO 2005/019836 so as to allow the vessel to be moved in an apparatus adapted to carry out reactions automatically.

At the lower end of the sample receiving portion (2), the vessel terminates in a capillary tube (3) which is sealed at the lower end by a transparent seal (4), so as to form an elongate thin reaction vessel, which can contain relatively small sample (7) within the capillary section.

An aluminium coating layer (5) surrounds the capillary tube (3) and is in close thermal contact with it. This coating layer (5) acts as a thermal shunt, which is able to rapidly dissipate thermal gradients which build up along the tube (3) and thus within a sample (7) which is subject to heating and/or cooling. The outer surface of the aluminium coating layer (5) has been anodised so as to produce an insulating layer thereof. In an alternative embodiment however, the outer surface of the aluminium coating layer (5) is coated with parylene to provide electrical insulation.

An ECP layer (8) completely encases the aluminium coating layer (5) as well as the sides of the lower seal (4) and the base of the upper portion (2). Is it provided with upper and lower ridges (9, 10) respectively which can accommodate upper and lower annular electrical connectors (11, 12) respectively. Each electrical connector (11,12) is provided with a number of inwardly projecting barbs (13) (FIGS. 3 and 4) which are able to pierce the surface of the ECP to ensure that electrical contact is made with the body of the ECP.

In use, this particular vessel can be loaded with sample and PCR reagents, as described in WO 2005/019836. A prepared sample (7) to which has been added all the reagents necessary for carrying a PCR reaction is placed in the upper portion (2) and if necessary a cap (not shown) is placed over open end. The entire vessel is then then centrifuged to force the sample (7) into the capillary tube (3) section of the vessel.

In an alternative embodiment however, the sample and the reagents may be loaded directly into the capillary tube (3) using a specifically designed fine tipped pipettor, and with accompanying close control of pipettor removal, as described and claimed in a copending British patent application of the applicants of even date to the present application.

The vessel is then suitably positioned in an apparatus (FIG. 5) able to accommodate it such that the connectors (11, 12) are connectable to an electrical supply but seat on a pair of supports.

A ring of a solid foam insulator material (14) (a polyurethane engineering foam) is provided in contact with the lower edge portion of the upper electrical contact (11). The lower electrical contact (12) is held on a shaped support (15), also of the solid foam insulator material.

The solid foam insulator material is arranged to minimise heat transfer from the electrical contacts, but does not interfere with the contact to the electrical supply (not shown).

A ring (16) of solid insulator material such as Delrin is provided adjacent the ring of solid foam insulator material (14) in contact with it. The ring (16) effectively surrounds the rest of the upper electrical contact (11) but is not in direct contact with it. As a result, it acts as an insulator from environmental effects coming from above, but does not act as either a heat sink or heat source in relation to the electrical contact itself.

Similarly, the shaped support (15) of a solid foam insulator material is itself supported on a ring (17) of solid insulator material such as Delrin so as to provide similar protection for the lower electrical contact (5). The solid insulator rings (16, 17) are provided with conduits for electrical connection and spring-mounted in housing (18).

In use, the solid insulator rings (16, 17) combined with the apparatus in which the vessel is held define a chamber for the tube (2) that is effectively isolated from the environment. However, the electrical contacts themselves are in contact with the solid foam material of low thermal mass.

When arranged in this way, the apparatus could be utilised in a polymerase chain reaction in a far more effective and reliable manner, as compared to devices which had no or alternative arrangements of insulator material.

The connectors (11,12) are then connected to the electrical supply, which is controlled, suitably automatically, to pass current through the ECP layer (5) so it rapidly progresses through a series of heating and cooling cycles, ensuring that the sample is subjected to similar cycling conditions. This will allow the sample (7) to be subjected to a PCR reaction.

Where a real-time monitoring system is included in the sample (7), the progress of the PCR can be monitored through the seal (4) using conventional methods.

As a result, rapid PCR can be achieved. The presence of thermal gradients within the vessel (1) and therefore the sample (7) is reduced by the measures taken, including in particular the presence of the aluminium coating layer (5) which acts as a thermal shunt. Thus reliable and reproducible results may be achieved.

During thermal cycling of the sample vessel in the PCR process, temperature gradients can arise along the length of the vessel. These temperature gradients typically arise due to the influence of the electrical connections, the temperature being higher in the region adjacent to the electrical connections during heating due to conduction. The applicants have discovered that the thermal gradients can be minimised by tapering the ECP coating of the sample vessel.

FIG. 6 illustrates the sample vessel of FIGS. 1 and 2. As previously described the sample vessel has a lower portion comprising a capillary which is coated in ECP. The capillary is 11.6 mm and FIG. 6 shows the radial thickness of the ECP at three positions along its length. The ECP tapers from an area of 3.12 mm at the top, 2.78 mm in the middle and 2.51 mm at the bottom.

FIGS. 7 and 8 show experimental data measuring the internal temperature of three sample vessels, having different radial thicknesses of ECP at the tip. The three sample vessels had uniform thickness of ECP along the length, apart from the tip 27, which had different adjustment to the tip diameter.

FIG. 7 illustrates the mean, tip and centre internal capillary temperatures at a thermopile setting of 43° C.

FIG. 8 illustrates the mean, tip and centre internal capillary temperatures at a thermopile setting of 63° C.

Both sets of results show that when the tip diameter was reduced, causing a tapering of the ECP, the different between the temperature at the tip and centre was also reduced.

FIG. 9 shows the effect of tapering the ECP on the temperature gradient inside the capillary, showing results for thermopile settings at both 43° C. and 63° C. It can be seen that reducing the tip diameter significantly reduces the temperature difference between the tip and the centre and that the effect is increased for higher temperatures.

This effect of minimising temperature gradients by altering the ECP profile can be further enhanced by the use of a layer of highly thermally conducting material, for example aluminium as described in the previous embodiments.

The applicants have discovered that the profile of ECP can be adjusted to increase the thermal gradient. FIG. 9 shows that the temperature gradient inside the capillary is increased when the tip diameter is increased.

A temperature gradient caused by profiling ECP can be created as described in the following example.

EXAMPLE

A 1 mm thick sheet of ECP (carbon black filled polyethylene) was cut into the shape of an elongated equilateral triangle with the tip cut off, having dimension of 90 mm long, 20 mm wide at the base and 4 mm wide at the tip. This was connected to a laboratory power supply using crocodile clips that were attached at either end.

A ruler was used to measure 10 mm steps (from the narrow end to the wide end) starting at 10 mm from the narrow end and finishing at 70 mm from the narrow end. The ECP was placed on a foam block and temperature at each step was measured by placing a thermocouple probe on the surface with a small pad of foam on the top.

The temperature measurements are shown in FIG. 10. As can be seen, a temperature gradient is formed between the higher temperature at the narrow end and the lower temperature at the wider end.

1. A reaction vessel heating system, which comprises electrically conducting polymer, arranged to act as a heating element for a reaction vessel, wherein the profile of the electrically conductive polymer differs in different regions of the vessel so as to control thermal gradients therealong. 2. A reaction vessel heating system according to claim 1 wherein the profile of the ECP is arranged to have the effect of reducing thermal gradients therealong. 3. A reaction vessel heating system according to any of claims 1 and 2 wherein at least one wall of said vessel comprises a highly thermally conducting layer. 4. A reaction vessel according to claim 3 wherein the highly thermally conducting layer is a metallic layer. 5. A reaction vessel according to any of claims 3 or 4 further comprising an inner non metallic layer. 6. A reaction vessel according to any of claims 4 or 5 wherein the electrically conductive polymer is insulated from the metallic layer by means of an insulating layer therebetween. 7. A reaction vessel according to claim 6 wherein the insulating layer is a layer of anodised aluminium, or is a polymer layer. 8. A reaction vessel according to claim 7 wherein the polymer layer comprises parylene or a derivative thereof. 9. A reaction vessel according to any preceding claim which is an elongate vessel. 10. A reaction vessel according to claim 9 which comprises a tube which is sealed at one end, wherein the end is of a transparent material 11. A reaction vessel according to any one of claims 9-10 which comprises a capillary vessel or a flattened capillary vessel. 12. A reaction vessel according to any of claims 9-11 wherein the radial depth of ECP material at the tip of the vessel is less than the radial depth of ECP material at the centre of the vessel. 13. A reaction vessel according to any of claims 1-12 wherein the vessel is a thermal cycling vessel. 14. A reaction vessel according to any of claims 1-113 wherein the vessel is a PCR reaction vessel. 15. A reaction vessel heating system according to claim 1 wherein the ECP profile is arranged to have the effect of increasing thermal gradients. 16. A reaction vessel according to claim 15 wherein the vessel is a culture vessel for the culture of biological materials. 17. A reaction vessel according to claim 16 wherein the vessel is one of a petri dish, cuvette, chemostat, shake flask, universal container, bijou. 18. A method for carrying out a chemical or biochemical reaction which requires at least one heating step, said method comprising placing chemical or biochemical reagents into a reaction vessel according to any one of claims 1 to 17 and heating said reagents so as to bring about a chemical or biochemical reaction. 19. A method according to claim 18 wherein the reaction requires thermal cycling. 20. A method according to claim 19 wherein the reaction is a polymerase chain reaction. 21. A method for mixing reagents in a vessel, said method comprising placing said chemical or biochemical reagents in a reaction vessel according to any of claims 15-17 and heating the heating the vessel so as to bring about a temperature gradient to create convection.


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stats Patent Info
Application #
US 20110065150 A1
Publish Date
03/17/2011
Document #
File Date
11/21/2014
USPTO Class
Other USPTO Classes
International Class
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Drawings
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Polymerase Chain Reaction


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