Detailed Description
One aspect of the present invention is a method for nucleic acid amplification comprising
a) Providing a porous matrix configured to provide compartments,
b) adding a sample comprising nucleic acids and an amplification mixture to the porous matrix,
c) exposing the porous matrix to a temperature cycle,
wherein the nucleic acid amplification occurs within the pores of the porous matrix.
The method of the invention can be performed in at least 2 different ways with respect to the amplification mixture required for nucleic acid amplification. Those skilled in the art know that enzymes, primers and nucleotides along with appropriate buffers, solvents and/or detergents are required to perform nucleic acid amplification.
Thus, in a preferred method according to the invention, the amplification mixture comprises enzymes, primers, nucleotides and buffers.
For nucleic acid amplification to occur within the pores of a porous matrix, the porous matrix must be in physical contact with the sample comprising nucleic acid and the amplification mixture. This can be ensured, for example, by dipping the porous substrate into a solution comprising the nucleic acid-containing sample and the amplification mixture, or by spotting or pipetting the nucleic acid-containing sample and the amplification mixture onto defined areas of the porous substrate. The advantages of the spotting or pipetting embodiment are of course that smaller amounts of sample and reagents are required and that more than one sample can be applied to 1 porous matrix. It is possible to add the nucleic acid containing sample and the amplification mixture to the porous matrix sequentially or in one step by several techniques, such as pipetting, ink jet needle printing or microchannel deposition.
In another preferred method according to the invention, said porous matrix in step a) is provided with at least one attached primer and/or said amplification mixture comprising an enzyme, nucleotides and a buffer.
In this embodiment of the invention, the primers required for the amplification reaction are already present on the porous matrix prior to addition to the amplification mixture. The attachment of the primers to the surface of the porous matrix is preferred. As mentioned above, the surface of the porous matrix summarizes all interfaces of the porous matrix with the surrounding environment, in other words the exterior of the matrix and the interior of the pores.
The attached primers are primers that bind to the surface of the porous matrix. Within the scope of the present invention are all kinds of bonds known to the person skilled in the art. Examples are covalent bonds such as silane, amide or epoxide bonds, coordinate bonds such as between a His tag and a chelating agent, bioaffinity (bioaffine) bonds such as the biotin/streptavidin bond. Alternatively, the binding of the primer to the porous matrix may be physisorption. In this embodiment, the primer is simply applied to the porous matrix by, for example, spotting or pipetting the primer to the matrix followed by evaporation of the solvent.
In a more preferred method according to the invention, the at least one attached primer is synthesized on a porous substrate.
In another more preferred method according to the invention, the at least one attached primer is a synthetic primer spotted on the porous substrate.
There are mainly 2 different strategies to provide a porous substrate with at least one attached primer, i.e.the binding of the whole primer to the substrate (off-chip synthesis) or the synthesis of the primer on the substrate (on-chip synthesis).
For off-chip synthesis, the porous substrate may be immersed in a solution containing the primers or by, for example, spotting or pipetting the primers to defined areas of the porous substrate. If not only physisorption is required, the subsequent coupling is dependent on the matrix material used and the binding moiety of the primer to be achieved, and several alternatives are known to the person skilled in the art. Possible surface modifications may be epoxy functionalization, such as epoxysilane derivatives, or aldehyde functionalization, or hydroxyl functionalization, or thiol (thiol) functionalization, or amino functionalization, such as aminopropyltriethoxysilane, or multifunctional amino coatings (see, for example, the commercial products from Schott Nexterion). For covalent coupling of the spotted primers to the surface different techniques can be used, such as photochemical coupling via e.g. UV-mediated cross-linking, wet-chemically assisted coupling using suitable reagents, electrochemically mediated coupling such as redox coupling or cross-coupling via e.g. Diels-Alder reaction.
During on-chip synthesis, primers are synthesized from single nucleotides, oligonucleotides or polynucleotides (referred to throughout the invention as nucleotide building blocks) in more than one step on a porous substrate. Each step of this procedure is referred to throughout the present invention as a synthesis cycle.
Preferably, the synthesis or coupling of the primers on the porous substrate throughout the present invention is performed by electrochemical procedures. In order to achieve electrochemical production of such porous matrices with attached primers, the porous matrix and/or the nucleotide building blocks must have binding sites protected by protecting groups, which are however electrochemically unstable. Thus, each synthesis cycle of electrochemical production involves at least one situation in which an electrical potential is applied to the porous substrate, electrochemically deprotecting those protecting groups of the binding sites that are electrochemically unstable at the applied potential and that are located on certain parts of the porous substrate and/or on certain nucleotide building blocks that have been attached to the porous substrate. Deprotection of a protecting group can occur by cleavage of the entire protecting group, cleavage of portions of the protecting group, or by conformational changes within the protecting group. Electrochemical deprotection of electrochemically labile protecting groups includes direct deprotection via an applied potential and deprotection via mediators generated on certain electrode surfaces of an electrode array due to the applied potential. After deprotection of certain protecting groups, a single nucleotide, oligonucleotide or polynucleotide may be bound to the deprotected binding site.
Furthermore, electrodes are necessary to apply the potential to achieve electrochemical production of such porous matrices with attached primers. Preferably, the electrodes are arranged in the form of an electrode array comprising a solid support and an arrangement of more than one individual electrode. Any material may be used for these individual electrodes, i.e. metallic or semiconducting, in so far as it has a suitable conductivity and in so far as it is electrochemically stable over a certain potential range. For the solid support of the individual electrodes, any material can be used in terms of having the property of avoiding short circuits between the individual electrodes.
The arrangement of the individual electrodes is designed such that each electrode is a selectively addressable electrode. Thus, the design of the arrangement of the individual electrodes provides the option to address a certain number of electrodes simultaneously in a group by means of a potential or to address each electrode independently.
Each electrode of the electrode array defines a region on the porous substrate in which an electrochemical reaction can occur as a result of an electrical potential applied across the electrode. Thus, each electrode corresponds to an individual spot on the porous matrix, and each individual spot contains certain primers after the electrochemical student, resulting in an array of primers that can be defined by the production procedure.
Throughout the present invention, preferred protecting groups are acid-labile protecting groups, preferably 9- (9-phenyl) xanthenyl (pixyl) or trityl, most preferably 4, 4' -Dimethoxytrityl (DMT) or 4-monomethoxytrityl (MMT), or base-labile protecting groups, preferably acetylpropionyl (levulinyl) or silyl, most preferably tert-butyldimethylsilyl (TBDMS) or tert-butyldiphenylsilyl (TBDPS).
In a more preferred method according to the invention, the attached primer is cleaved from the porous matrix before performing the temperature cycling.
In this preferred method according to the invention, the primers coupled to the porous matrix may be released prior to nucleic acid amplification. Therefore, the coupling of primers to porous matrices must be unstable under certain conditions. Cleavage of the primers to the porous matrix can be performed using an electrical potential, irradiation (e.g., UV light), heat, or chemical treatment. Possible cleavable linkers for the primer are base labile moieties such as succinyl, oxalyl or hydroquinone linkers (Q linkers), or photolabile moieties such as 2-nitrobenzyl-succinyl or veratrole-carbonate linkers, or linkers cleavable under reducing conditions such as thio-succinyl linkers, or acid labile moieties such as trityl derivatives, e.g. 4, 4' -dimethoxytrityl derivatives.
It should be noted that nucleic acid amplification can be performed within the pores of the porous matrix with cleaved primers and with attached primers. Nucleic acid amplification occurs during a temperature cycle comprising a heating, cooling phase and a thermostating phase, while the temperature at the beginning of 1 cycle is the same as the temperature at the end of the cycle. After the final temperature cycle, an amount of amplified nucleic acid is present within the pores of the porous matrix. There are mainly 2 different procedures for detecting or analyzing amplification products within the porous matrix, depending on the needs of the user.
In another preferred embodiment of the present invention, the amplified nucleic acids are extracted from the porous matrix by centrifugation.
With the porous matrix according to the invention it is possible to remove the amplification product for external analysis, for example by means of gel electrophoresis, hybridization assays or mass spectrometry.
If a compartment-free unstructured porous matrix is used, the entire matrix can be placed in a centrifugation vessel to extract the amplified nucleic acids. If a structured porous matrix with compartments is used, one must ensure that the amplified nucleic acid from each compartment is collected in a separate container. This can be achieved by using a microtiter plate which is adjusted to the size and distribution of the compartments of the porous matrix in such a way that each compartment is above a well of the microtiter plate if the porous matrix is placed above said microtiter plate.
Alternatively, the porous matrix may be cut into several parts each comprising only 1 compartment. Thereafter, each of the porous matrix portions may be placed in a separate centrifugation vessel for extraction of the respective nucleic acid.
Furthermore, extraction of the amplified nucleic acids can be done by applying a pressure difference (e.g. vacuum) or a liquid that blots the membrane (e.g. with a pipette).
A preferred embodiment of the invention is a method wherein the amplified nucleic acids are detected within said porous matrix.
Alternatively, the amplification products can be detected directly in a porous matrix, and the detection based on fluorescence is preferred, since standard techniques for analyzing PCR amplification are based on fluorescent dyes, such as intercalating dyes or labeled hybridization probes. In this embodiment, the amplification mixture comprises a fluorescent compound for detecting the respective amplification product. For example, the amplification mixture may comprise several labeled hybridization probes selected from the group consisting of FRET hybridization probes, TaqMan probes, molecular beacons, and single-labeled probes. Alternatively, dsDNA binding fluorescent entities such as SYBRGreen may be used, which fluoresce only when bound to double stranded nucleic acids.
Furthermore, detection of amplified nucleic acids within the porous matrix may be performed using electrochemical techniques. In this embodiment, the electrode is placed under a porous matrix to apply an electrical potential and the hybridization probe is labeled with an electrochemical moiety such as a ferrocene (ferrocen) derivative or osmium complex.
Furthermore, detection of amplified nucleic acids within the porous matrix may be performed using chemiluminescence techniques.
A more preferred embodiment of the invention is a method wherein the amplified nucleic acids are detected by fluorescence, preferably in real time.
If the amplification mixture comprises fluorescent probes, it is preferred to monitor not only the fluorescence of one amplification at the end of the amplification but also at least once per amplification cycle. In other words, real-time PCR is preferably performed within the pores of the porous matrix.
The methods of the invention provide a porous matrix that is configured to provide compartments.
Throughout the present invention, compartments are regions of a porous matrix separated from each other by impermeable borders (boarders). In other words, the impermeable border around the porous matrix compartments avoids liquid exchange between adjacent compartments. Thus, it is possible to perform several assays in parallel using only 1 porous matrix, since the impermeable border avoids cross-talk between compartments.
It should be noted that throughout the present invention, the structure of the porous matrix providing the compartments optionally comprises not only the compartments themselves, but also a channel structure for liquid communication between the compartments and the outside of the membrane. Furthermore, it is possible to provide a porous matrix with a channel structure connecting 2 or more compartments, if this is required for certain applications. The fluid may penetrate the channel structure, for example by gravity, capillary force, pressure or centrifugation.
Another more preferred embodiment of the invention is a method wherein individual nucleic acid amplifications are performed in each of said compartments.
Using a structured porous matrix, it is of course possible to perform individual nucleic acid amplifications in each compartment, while there are mainly 2 different alternatives for this purpose.
In a more preferred method of the invention, the individual nucleic acid amplifications are the same or different.
In another more preferred method of the invention, the different nucleic acid amplifications are based on different samples and/or different primers within the compartments.
One reason for performing identical nucleic acid amplification in all compartments may be to produce increased reliability with respect to the amplification results. Performing different nucleic acid amplifications in each compartment increases the throughput of sample analysis. Different nucleic acid amplifications can be established by analyzing different primers for the same sample in each compartment, or by analyzing different samples through a number of equivalent compartments. Preferably, a porous substrate is provided having compartments each having one or more different primers attached to a pore surface within the compartment.
In a preferred embodiment of the method according to the invention, the porous matrix has a size of 1x 10-2cm2-2 x 102cm2Area sum of 1x 10-2cm-0.5cm height, preferably 1x 10-1cm2-1 x 102cm2Area of (3 x 10)-2A height of cm-0.3cm, most preferably 1cm2-1 x 102cm2Area of (2) and 5x 10-2cm-0.2 cm.
Regarding the size and height of the porous matrix, several aspects must be considered. First, the area of the porous matrix must be large enough to fully enable the formation of the compartments and to enable the arrangement of the desired number of said compartments. Thus, the area of the porous matrix also depends on the expected size of each compartment and the surrounding barrier. On the one hand, the height of the porous matrix must be large enough to provide a volume of compartments to perform PCR amplification and, on the other hand, thin enough to enable fluorescence detection throughout the entire volume if fluorescence techniques are used to analyze the amplification products.
In another preferred embodiment of the method according to the invention, the porous matrix has at least 2 compartments, preferably 2-1 x 106A compartment, most preferably 1x 102-1 x 105And (4) a plurality of compartments.
Another preferred method according to the invention is a method wherein said compartments are provided by chemical functionalization of said porous matrix.
There are several possibilities to provide a porous matrix with compartments. The phrase chemical functionalization outlines all procedures for chemically modifying the surface properties of a porous substrate. It is possible to modify the surface properties of the porous matrix by wet chemical treatment, photochemical treatment, ion bombardment, temperature or by electrochemistry. It should be noted that chemical functionalization of the porous matrix can be performed directly or indirectly by the above-described technique, wherein the above-described technique only performs surface activation, such that further moieties can thereafter bind to the activated binding sites.
Another preferred process according to the present invention is a process wherein said chemical functionalization is carried out electrochemically.
The use of electrochemical methods is preferred, since the surface modification of the porous matrix can be performed in a controlled manner using an electrode array as explained above.
For nucleic acid amplification in a compartment, it is preferred that the compartment is hydrophilic embedded in a hydrophobic surrounding environment. In general, the procedure for creating the hydrophilic/hydrophobic pattern must distinguish between different regions of the porous matrix.
Techniques that enable such a mode to be generated are, for example, electrochemical by applying a certain current or voltage to certain areas of the porous matrix, photochemical by applying a certain wavelength of light to certain areas of the porous matrix, or spotting techniques by applying a volume of reagent to certain areas of the porous matrix.
The starting point for the construction of the porous matrix may be a pre-processed porous matrix with free functional moieties such as carboxyl, epoxy, aldehyde, hydroxyamino, or a pre-processed porous matrix with protected functional moieties (groups blocked by chemical residues as mentioned above for on-chip synthesis of primers), or a non-pre-processed porous matrix without functional groups.
With a pre-processed porous matrix with free functional groups, electrochemical, photochemical or spotting techniques must address certain areas of the porous matrix to attach hydrophilic or hydrophobic moieties.
With a pre-processed porous substrate with protected functional groups, electrochemical, photochemical or spotting techniques must address certain areas of the porous substrate to enable chemical reactions to be effected to attach or deprotect hydrophilic or hydrophobic moieties. For example, a porous substrate having functional moieties protected by hydrophobic groups may be deprotected to produce hydrophilic regions, and untreated regions will remain hydrophobic. The reverse process with functional moieties protected by hydrophilic groups can be used to generate patterns by cleaving the hydrophilic protecting groups. To create hydrophobic regions, it may be useful to couple additional hydrophobic residues to the deprotected regions after the deprotection.
With a non-pre-processed porous matrix that does not contain functional groups, hydrophilic/hydrophobic patterns can be created by modifying certain regions of the porous matrix with functional groups.
To create a hydrophilic/hydrophobic pattern, different groups can be used. For hydrophilic regions, hydrophilic groups such as hydroxyl, amino, carboxyl, thiol, phosphate are useful. For hydrophobic regions, hydrophobic groups such as cholesterol, carbon alcohols (e.g. dodecanol), trityl derivatives or palmitoyl are suitable. To enhance the hydrophilic/hydrophobic properties, multifunctional residues such as dendrimers or branched derivatives may also be used. Preferably, the hydrophilic/hydrophobic residues are covalently coupled to the porous matrix to provide sufficient stability for subsequent amplification reactions.
Further preferred is a method according to the invention wherein the compartments are provided by fluidic spotting.
A fluid suitable for constructing a porous matrix to provide compartments is a material dissolved in a solvent which evaporates at atmospheric pressure and thus forms a thin film of the material. An example of such a strategy is a solution of polyvinyl chloride (PVC) in Tetrahydrofuran (THF).
It should be noted that it is possible to provide a porous matrix with more than one type of primer molecules/compartments. This can be accomplished by using compartments with orthogonal protecting groups for generating primer arrays, whereas the orthogonal protecting groups are at least 2 different protecting groups that are unstable under different conditions, such as different potentials, acid/base lability or any other combination of electrochemical, wet chemistry and photochemical. The use of such orthogonal protecting groups, for example for protecting binding sites of a porous matrix, provides the opportunity to generate a mixture of more than one type of primer in 1 individual compartment of the porous matrix. At least 2 different protecting groups may be provided, each as an individual surface modification or as a single branched surface modification comprising 2 or more of said different protecting groups.
A preferred method according to the invention is a method wherein an additional prehybridization step is performed before exposing the porous matrix to temperature cycling and before optional cleavage of primers to the porous matrix.
Providing an array of primers has the further advantage that a prehybridization step can be performed before the amplification reaction to accumulate certain nucleic acids of the applied sample on the corresponding compartments of the structured porous matrix. This prehybridization step is a hybridization step that occurs if nucleic acids within the sample are contacted with covalently bound primers of the respective regions. In other words, each target nucleic acid within the sample finds an attached primer with a complementary sequence prior to a subsequent amplification reaction. Due to this pre-hybridization step, it is possible to detect much lower concentrations, since the detection limit is no longer dependent on the statistical distribution of each nucleic acid across all compartments of the array.
In a more preferred method according to the invention, the porous matrix is sealed to avoid cross-talk between compartments.
Figure 1 shows a schematic diagram illustrating one embodiment of a porous matrix that is sealed to avoid cross-talk between compartments. If the porous matrix 1 is configured to provide compartments 2, it is important to avoid cross-talk not only within the porous matrix but also between compartments via its surroundings. Therefore, it is preferred to seal the porous matrix after application of the sample and/or amplification mixture and before PCR amplification. All kinds of seals 5, 6 for aqueous solutions may be known to the person skilled in the art throughout the present invention. Examples are plastic foils which may be adhered to the surface of the porous substrate, for example, or slides which may be pressed against the porous substrate by mechanical force to provide a water-tight contact. If the slide is used to seal a porous substrate, a hydrophobic slide (e.g., silanized glass) or an intermediate oil film is preferably used. Alternatively, the entire porous matrix may be immersed in an oil, such as PCR oil. Furthermore, evaporating the fluid thus forms a thin film on the surface, such as polyvinyl chloride (PVC) dissolved in Tetrahydrofuran (THF), suitable for sealing porous substrates. It should be noted that if fluorescence detection, e.g. PCR within a porous matrix, is required, optically transparent materials have to be used, and reversible sealing is necessary if subsequent extraction of amplified nucleic acids is to be expected.
In a preferred method according to the invention, a thermal substrate 6 is used to seal one side of the porous substrate. The thermal Base is a special heat pipe (heat pipe) in the form of a plate, which is used as thermal-BaseTMCommercially available from Thermocore (Lancester, USA). A heat pipe is a sealed vacuum vessel with an internal wick structure that transfers heat through evaporation and condensation of an internal working fluid. Typically ammonia, water, acetone or methanol are used, although special fluids are used for low and high temperature applications. As heat is absorbed on one side of the heat pipe, the working fluid is evaporated, creating a pressure gradient within the heat pipe. The vapor is forced to flow to the cooler end of the tube where it condenses, transferring its latent heat to the core structure and then to the ambient environment via, for example, a heat sink. The condensed working fluid returns to the evaporator via gravity or capillary action within the internal wick structure. Because heat pipes utilize the latent heat of the working fluid, they can be designed to maintain the composition near ambient conditions. The heat pipes may operate in any orientation, although they are most efficient when the condensed fluid operates by gravity.
Thus, the use of a thermal substrate for sealing one side of the porous matrix has an additional advantageous effect in terms of thermal cycling, which is necessary for PCR amplification within the porous matrix. Some more details about embodiments of the invention that include a thermal base may be found later in the specification.
It should be noted that if the porous matrix is configured to provide compartments and channel structures, the order of sealing the porous matrix after application of the sample and amplification mixture may be varied. In this case, the porous matrix may be sealed, for example, after the primers have been attached to the pores within the chamber. Thereafter, the amplification mixture and the sample are applied to the compartments via the channel structure to perform the amplification reaction.
Another procedure for providing compartments within a porous matrix is to use mechanical pressure to partially compress the matrix, thereby causing the pores to be closed or minimized and diffusion of the liquid to be hindered.
Furthermore, the porous matrix may be configured to provide compartments by using a temperature or laser treatment that partially melts the porous matrix, such that the pores of the porous matrix become closed in the treated area.
In another preferred method according to the invention, the porous substrate is a glass wool, an organic polymer such as cellulose, or an inorganic polymer such as nylon, polyester, polypropylene (PP), Polyethylene (PE), polyethylene terephthalate (PET), Polyacrylonitrile (PAT), polyvinylidene fluoride (PVDF), or polystyrene.
Furthermore, other materials such as glass, metal oxides or silicon derivatives are suitable for the present invention insofar as they are processed in such a way that they provide pores in which nucleic acids can be amplified.
Another aspect of the invention is a porous matrix for nucleic acid amplification comprising
a) A plurality of compartments for performing individual nucleic acid amplifications in parallel,
b) pores that enable diffusion of nucleic acid molecules and polymerases for nucleic acid amplification within the pores of the porous matrix, and
c) at least one primer attached to a surface of the porous substrate.
The primers can be attached to the porous matrix by any procedure known to those skilled in the art. Examples are covalent bonds such as silane, amide, aldehyde or epoxide couplings, cross-couplings via for example Diels-Alder reaction, coordination bonds such as between a His tag and a chelating agent, bioaffinity bonds such as biotin/streptavidin bonds. Alternatively, the binding of the primer to the porous matrix may be physisorption. In this embodiment, the primer is simply applied to the porous matrix by, for example, spotting or pipetting the primer to the matrix followed by evaporation of the solvent.
In a preferred porous matrix according to the invention, the primers are covalently attached to the porous matrix.
The porous matrix according to the invention has compartments for performing a number of individual nucleic acid amplifications in parallel.
Different possibilities and requirements regarding porous matrices with compartments with or without channel structures have been explained previously. It should be noted that if the different nucleic acid amplifications are performed in compartments of the porous matrix, it is preferred to seal the porous matrix after sample, primer and/or probe loading and before the amplification reaction. If a channel structure is provided, the porous matrix may alternatively be sealed prior to loading of the sample and amplification mixture.
In another preferred porous matrix according to the invention, said compartments are defined by a chemical barrier, preferably said chemical barrier is a chemical functionalization of said porous matrix.
As previously mentioned, one possibility for constructing the porous matrix is a chemical functionalization of the material of the porous matrix. For example, certain portions of a hydrophilic porous matrix may be altered such that it is hydrophobic thereafter. In other words, the functionalized hydrophobic portion of the hydrophilic porous matrix forms a chemical barrier with respect to aqueous solutions.
In a more preferred porous matrix according to the invention, said chemical functionalization is an electrochemical functionalization.
In another preferred porous matrix according to the invention, the compartments are defined by fluidic spotting.
The present invention has been outlined previously with respect to alternatives of electrochemical functionalization and fluidic spotting to construct porous matrices to provide compartments with or without channel structures.
Another preferred porous matrix according to the invention is a matrix wherein each compartment has the same or different attached primers.
In general, compartmentalization of a porous matrix is provided to perform multiple different amplification reactions in parallel, and there are mainly 2 different alternatives, i.e. the same primer set and different samples or different primer sets and the same sample. Thus, a porous matrix with the same set of primers in each compartment can be provided for screening multiple samples, or a porous matrix with different primers in each compartment for screening samples for several components.
Another aspect of the invention is a multiwell plate for nucleic acid amplification, wherein each well of said multiwell plate comprises a porous matrix according to the invention, such that nucleic acid amplification takes place within said well of said porous matrix.
The use of a multi-well plate for processing a plurality of porous substrates has the following advantages: such devices are applicable to many commercial apparatus such as block cyclers (blockcyclers) to perform amplification reactions in a controlled and highly parallel manner. In addition multi-well plates are compatible with techniques that increase throughput for screening purposes, such as automated pipetting using robotic instruments, analytical techniques with standard detection instruments.
Another aspect of the present invention is a kit for nucleic acid amplification comprising
a) The porous matrix according to the invention, and
b) the mixture is amplified.
Throughout the present invention, the amplification mixture comprises all compounds necessary to perform a nucleic acid amplification reaction in the form of a Polymerase Chain Reaction (PCR), i.e. a thermostable DNA polymerase, at least one nucleic acid compound, deoxynucleotides, a nucleic acid having at least one type of divalent cation, preferably Mg2+The buffer of (4). In addition, the amplification mixture may contain, for example, synthetic peptides or other PCR additives with divalent cation binding sites for "hot start" PCR.
In a preferred kit according to the invention, the amplification mixture comprises enzymes, primers, nucleotides and buffers.
As thermostable polymerases, various enzymes can be used. Preferably, the thermostable DNA polymerase is selected from the group consisting of Aeromonas sobria (Aeropyrum pernix), Archaeoglobus fulgidus (Archaeoglobus fulgidus), Thiobacococcus species (Desulfococcus sp.) Tok, Methanobacterium thermonatum (Methanobacterium thermoautotrophicum), Methanococcus species (Methanococcus sp.), Methanococcus species (e.g.Methanococcus jannaschii, Methanococcus wolfei (voltae)), Methanothermus ferus (Methanobacterium perus pervivus), Pyrococcus species (Pyrococcus) such as Pyrococcus furiosus (furiosus), Methanococcus jannaschii (GB-D, Pyrococcus woeseii), Pyrosylvii, horikoshii, KOD, Deep Vent, Prooft, Pyromysi, Pyrococcus thermoneticus (Pyrococcus flavus), Thermoascus species (Thermoascus sp.), Thiocarpus thiopicus, Pyrococcus species (Thermoascus), Pyrococcus furiosus, Pyrococcus species (Thermoascus, Thermoascus species (S. sulphureus), Pyrococcus 3, Thermoascus species (S., The species JDF-3, gorgon ariius, TY), Thermoplasma acidophilum (Thermoplasma acidophilum), Thermus Africa (Thermosipho africana), Thermotoga sp (e.g., Thermotoga maritima (maritima), Thermotoga neapolitana (neapoliana)), Methanobacterium thermonatum, Thermus species (Thermus) (e.g., Thermus aquaticus (aquaticus), Thermus brockius (brockianus), Thermus filamentous Thermus (filformis), Thermus flavus (flavus), Thermus lactis (lactis), Thermus rubrus (rubens), Thermus rubrus (ruber), Thermus thermophilus (thermophyllus), ZO5, or Dynazyme). Also within the scope of the present invention are mutants, variants or derivatives, chimeric or "Fusion-polymerases" thereof, such as Phusion (Finnzymes or New England Biolabs, Cat. No. F-530S) or iPof (Biorad, Cat. No. 172-5300), Pfx Ultima (Invitrogen, Cat. No. 12355012) or HerculaseII Fusion (Stratagene, Cat. No. 600675). Furthermore, the composition according to the invention may comprise a blend of one or more of the above mentioned polymerases.
In one embodiment, the thermostable DNA polymerase is a DNA-dependent polymerase. In another embodiment, the thermostable DNA polymerase has additional reverse transcriptase activity and can be used for RT-PCR. An example of such an enzyme is Thermus thermophilus (Roche Diagnostics Cat. No.: 11480014001). Also within the scope of the invention are blends of one or more of the polymerases compiled above with retroviral reverse transcriptases, e.g., polymerases from MMLV, AMV, AMLV, HIV, EIAV, RSV, and mutants of these reverse transcriptases.
The concentrations of DNA polymerase, deoxynucleotides, and other buffer components are present in standard amounts, the concentrations of which are well known in the art. Mg (magnesium)2+The concentration may be between 0.1mM and 3mM and is preferably adapted and experimentally optimized. However, since the concentration optimum generally depends on the actual primer sequence used, it cannot be theoretically predicted.
The at least one nucleic acid compound of the amplification mixture comprises at least one pair of amplification primers to perform a nucleic acid amplification reaction.
Furthermore, the amplification mixture may comprise fluorescent compounds for real-time detection of the respective amplification products, and respectively 2-6 differently labeled hybridization probes, which are not limited to but selected from the group consisting of FRET hybridization probes, TaqMan probes, molecular beacons, and single labeled probes. Alternatively, such an amplification mixture may comprise a dsDNA-binding fluorescent entity, such as SYBRGreen, which fluoresces only when bound to double stranded DNA.
In addition, the amplification mixture may be adapted to perform single-step RT-PCR and comprise a blend of taq dna polymerase and a reverse transcriptase, such as AMV reverse transcriptase. In further exemplary embodiments, such amplification mixtures are particularly adapted to perform single-step real-time RT-PCR and comprise a nucleic acid detection entity such as SYBR Green or a fluorescently labeled nucleic acid detection probe.
In another preferred kit according to the invention, at least one primer is attached to the surface of the porous matrix and the amplification mixture comprises an enzyme and nucleotides.
In this embodiment of the kit, the primers have been attached to the surface of the porous matrix and thus the amplification mixture must not comprise primer molecules.
Another aspect of the present invention is a system for nucleic acid amplification comprising
a) The porous matrix according to the invention, and
b) a thermal cycler.
Throughout the present invention, the thermocycler summarizes all components necessary to perform thermal cycling with the porous matrix. The thermal cycler contains at least one heat pump, such as a Peltier element, to increase the temperature of the porous substrate, a heat sink to dissipate heat during cooling of the porous substrate, and a control unit to control said simultaneous thermal cycling of the various samples. As previously mentioned, it is preferred to provide an additional thermal base between the porous substrate and the heat sink to increase the speed and accuracy of the temperature change, as well as to provide a uniform heating/cooling procedure across the entire cross-sectional area of the porous substrate.
In a preferred system according to the invention, the thermocycler comprises at least one heat pump, a heat sink and/or a control unit.
In another preferred system according to the invention, the thermal cycler includes an illumination means and a detection means.
It is preferred to provide the system with additional detection means to analyze the amplification results directly on the porous matrix. Preferably the detection means is a fluorescence detector, since standard techniques for analysing PCR amplification are based on fluorescent dyes, such as intercalating dyes or labelled hybridisation probes. If the amplification results should be analyzed by fluorescence techniques, the amplification mixtures of the invention further comprise a fluorescent probe. Since fluorescence technology does require light for excitation of the fluorescent dye, a preferred system according to the invention further comprises illumination means.
Depending on the size of the cross-sectional area of the porous matrix, the fluorescence detector and the illumination means must meet specific requirements. If the porous matrix of the invention has compartments distributed over its cross-sectional area, one has to ensure that the compartments in the center of the porous matrix and the compartments at their boundaries obtain the same illumination and that the fluorescence intensities are recorded in a comparable manner. This may be achieved by using a fluorescence detector and/or an illumination means equipped with telecentric optics.
Within the scope of the invention, telecentric optics are optics with very small apertures and therefore provide high depth of focus. In other words, the telecentric light of the telecentric optics is quasi-parallel to the principal rays for all points across the object space that are parallel to the optical axis in the object space and/or the image space. Therefore, the quality of the illumination or detection tool using telecentricity in the object space is hardly noticeable for the distance of certain object points (object points) from the optics. The aperture of the telecentric optics is imaged at infinity. Furthermore, using telecentric light, good lateral homogeneity across the beam is ensured, and the locations at the center of the part are comparable to those at the part boundaries. Throughout the present invention, telecentric optics always include a field lens. In the context of the present invention, a field lens is a single lens closest to the objective lens, which determines the field of view of the instrument, contains one or more components (achromatic lenses), and contributes, in combination with further optical components of the apparatus, to telecentricity in the object and/or image side.
The field lens of the present invention transfers excitation light from the light source to the porous substrate and fluorescent signals from the porous substrate to the detector. This does not exclude the introduction of further optical components in the beam path, for example between the light source and the field lens, between the field lens and the detector or between the field lens and the porous substrate.
In another preferred system according to the invention, the nucleic acid amplification is real-time PCR.
If the system according to the invention is equipped with a fluorescence detector and illumination means, it is preferred to monitor not only the fluorescence of one amplification at the end of the amplification but also at least once in each amplification cycle. In other words, real-time PCR is preferably performed within the pores of the porous matrix.
Yet another preferred system according to the invention further comprises means for extracting amplified nucleic acids from said apparatus for nucleic acid amplification.
In another embodiment of such a system according to the invention, the system is provided with further means for extracting amplified nucleic acids from the porous matrix. In certain embodiments, it may be desirable to extract amplified nucleic acids from the porous matrix for subsequent analysis. Such external analysis is for example mass spectrometry or gel analysis.
In a more preferred system according to the invention, said means for extracting amplified nucleic acids is a centrifugation means.
The different procedures for extracting amplified nucleic acids from a porous matrix with or without compartments have been explained in detail previously.
The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made in the procedures set forth without departing from the spirit of the invention.
Example 1:
the use of electrochemical procedures and the assignment of labeled oligonucleotides to the matrix creates a hydrophilic/hydrophobic pattern on the porous matrix.
The preparation of a hydrophilic/hydrophobic pattern comprising 2 hydrophilic sites on a hydrophobic matrix was generated using the electrochemical procedure described in figure 3. In this example, the coupling of hydrophobic moieties to a hydrophilic functionalized porous matrix is described. For this experiment, a home-made reaction chamber was used containing an electrode array with 2 gold electrodes, an inorganic porous matrix, standard DNA synthesis reagents, phosphoramidites of hydrophobic moieties and a buffer solution that electrochemically generates an acidic medium on the activated electrodes.
A porous substrate (PE-Sinter membrane from Polyan, Berlin/Germany, pore size: 10 μm, thickness: 0.6 mm; packing density of hydroxyl groups: 1.7. mu. mol/cm)2) Is disposed in the reaction chamber proximate to the electrode. Since the porous matrix itself has only binding sites without any protecting groups, 5 '-DMT-T-3' -phosphoramidite is coupled to the porous matrix as a starting group. For this purpose, 5 '-DMT-T-3' -phosphoramidite together with an activator (Dicyanoimidazol) dissolved in acetonitrile) was filled into the chamber to react with the functional groups of the membrane.
The solution is thereafter removed and subjected to an oxidation step to oxidize the trivalent phosphorus molecules from the first coupling step to more stable pentavalent phosphorus molecules. The oxidizing solution is then rinsed from the reaction chamber and a capping step is performed to block all unreacted hydroxyl groups of the porous matrix from the first coupling step for further reaction. Thereafter, the capping solution was taken out and the chamber was filled with the buffer solution. To modify a porous matrix with hydrophilic spots and a hydrophobic surrounding (fig. 2a), the following procedure (illustrated in fig. 3) was used. An electrical potential (-300 μ a 60 sec) was applied to the 2 electrodes in sequence to cleave the protecting groups on those portions of the porous substrate that were in proximity to the activated electrode. Thereafter, the buffer solution was again rinsed out of the reaction chamber and a solution of chlorotrimethylsilane in pyridine was added for 10 minutes to react with the previously deprotected hydroxyl group. Thus, the hydroxyl group is blocked by a silyl group, which leads to the following conditions: with silyl-protected hydroxyl groups in positions above the electrodes and trityl-protected hydroxyl groups in positions next to the electrodes. Thus, an acidic solution of 3% trichloroacetic acid in dichloromethane (DMT-removal reagent, Roth, Karlsruhe/Germany, cat. No. 2257, 1) was added for 2 minutes to remove all remaining DMT groups next to the electrode. Thus, the deblocked hydroxyl group is now accessible to react with the hydrophobic moiety to create a hydrophobic region on the membrane. Thus, a cholesterol-phosphoramidite (a 0.1M solution of tetraethylene glycol cholesterol phosphoramidite in acetonitrile, chemnes corp., Wilmington, MA/USA, catalog number CLP-2704) is filled into the chamber with an activator to react on the deprotected binding sites of the porous matrix. After 2 minutes of incubation, the phosphoramidite solution was rinsed away and another oxidation step was performed to stabilize the trivalent phosphorus moiety. After the exchange of the oxidizing solution, an aqueous solution of ammonia was flushed into the reaction chamber for an incubation time of 60 minutes to release the silyl protecting groups from the position above the electrodes and deprotect all the phosphate protecting groups from the phosphate moieties. After some subsequent washing steps with acetonitrile and water, a hydrophilic/hydrophobic pattern is obtained with hydrophilicity above the electrodes and hydrophobicity next to the electrodes.
To modify a porous matrix with hydrophobic sites and a hydrophilic ambient (fig. 2b), a second matrix was prepared under similar conditions by using the following procedure, but with the opposite pattern. After first coupling to the substrate with 5 '-DMT-T-3' -phosphoramidite to produce an initial layer, the chamber was filled with buffer solution and a potential (-300 μ Α 60 seconds) was applied to 2 electrodes in sequence to cleave the protecting groups on those portions of the porous substrate in proximity to the activated electrode. Subsequently, a chamber was filled with cholesterol-phosphoramidite (0.1M solution of tetraethylene glycol cholesterol phosphoramidite in acetonitrile, chemconscorp., Wilmington, MA/USA, catalog number CLP-2704) and an activator to react on the deprotected binding sites of the porous matrix. After 2 minutes of incubation, the phosphoramidite solution was rinsed away and another oxidation step was performed to stabilize the trivalent phosphorus moiety. Thereafter the solution is removed and a capping reaction is performed to block all unreacted hydroxyl groups of the porous matrix from the cholesterol coupling step for further reaction. The capping solution was then removed and an acidic solution of 3% trichloroacetic acid in dichloromethane (DMT-removal reagent, Roth, Karlsruhe/Germany, Cat. No. 2257, 1) was added for 2 minutes to remove all remaining DMT groups next to the electrode. After the exchange of the acidic solution, an aqueous solution of ammonia was flushed into the reaction chamber for an incubation time of 60 minutes to deprotect all of the phosphate protecting groups from the phosphate moieties. Finally, some washing steps with acetonitrile and water were performed and a hydrophilic/hydrophobic pattern with hydrophobicity above the electrodes and hydrophilicity next to the electrodes was obtained.
After preparing 2 different functionalization patterns, the physical properties of the membrane were determined by applying it to 5' -Cy3- (T)15Aqueous solutions of the oligonucleotides were tested. After an incubation time of 5 minutes, the membrane was washed in 0.5 xsppe buffer solution and subsequently imaged with a standard digital camera.
The hydrophilic oligonucleotide is moved into the hydrophilic region of the membrane and tries to avoid the hydrophobic region. The panel in fig. 2 shows this behavior, with red oligonucleotides on the hydrophilic region above (fig. 2a) or beside (fig. 2b) the electrode, depending on the functionalization pattern.
Example 2:
porous matrix with hydrophilic/hydrophobic pattern in water
The membrane (PE-Sinter membrane from Polyan, Berlin/Germany, pore size: 7-16 μm, thickness: 0, 6 mm; packing density of hydroxyl groups: 0, 8. mu. mol/cm)2) According to the electrochemical procedure mentioned in example 1 (current: 300 μ A, deprotection time: 60 seconds) was prepared in a hydrophilic/hydrophobic mode with the cholesterol moiety above the electrode. After this preparation, the membrane was placed between 2 slides to form a reaction chamber, and water was flushed into this arrangement. Water diffuses into the hydrophilic portion of the membrane but does not enter the hydrophobic region. Figure 4 shows a picture of the thus treated membrane imaged on a lumimager instrument (Roche Applied Science, Mannheim, germany) in a 520nm channel.
Example 3:
changes in fluorescence intensity due to oligonucleotide hybridization in a porous matrix using SYBR Green I
2 membranes (PE-Sinter membranes from Polyan, pore size: 80-130 μm, thickness: 0.6 mm; packing density of hydroxyl groups: 1.3. mu. mol/cm) with plastic frames around the membrane edges to avoid liquid leakage were prepared2). For this purpose, a solution in Tetrahydrofuran (TH) is preparedF) And soaking the edges of the membrane into the solution. The organic solvent THF evaporated and PVC remained as a film on the membrane. Thereby trapping liquid that is distributed in the middle of such a membrane.
2 membranes were placed on slides and treated with 2 different end-point PCR solutions from a SYBR Green I assay performed with a LightCycler 2.0 instrument (Roche Applied Science, Mannheim, Germany). 2 different PCR reactions were performed using a SYBR Green I assay from Universal Probe library Control Set (Roche Applied Science, Mannheim, Germany, Cat. No. 04696417001; detailed sequence information is listed in the sequence section) in combination with Li lightcycler FastStart DNA MasterPLUS SYBR Green I (Roche Applied Science, Mannheim, Germany, Cat. No. 03515885), according to standard conditions described in the package insert.
The first PCR was a positive reaction using a primer pair (SEQ ID.1 and SEQ ID.2) and a synthetic template from the above-described kit (Control F from Universal Probe library Control Set (Roche applied Science, Mannheim, Germany)), and the other PCR was a negative Control experiment using the same primers but without template (so-called no-template Control, NTC). The end-point PCR solution was pipetted into the middle of 2 membranes. Positive PCR experiments were obtained on the membrane on the left side of fig. 5, and a second membrane negative control (NTC) on the right side of fig. 5. After dispensing the PCR solution, the membrane is then covered with another slide, and this reaction chamber is clamped with a clamp and sealed with an adhesive foil.
Thereafter, the fluorescence intensity of the slides was recorded in a Lumilmager instrument (Roche Applied Science, Mannheim, Germany) at 2 different temperatures in the 520nm channel, i.e.at room temperature or at 80 ℃. Due to the principle that the combination of double stranded DNA with the intercalating dye SYBR Green I leads to a fluorescent signal, there is a large amount of fluorescent signal for positive PCR experiments at room temperature, whereas only a small signal is obtained for control experiments (see fig. 5 a). At elevated temperature (80 ℃), the double stranded DNA melted and the signal of the SYBR Green I dye disappeared, so that 2 membranes all had less fluorescence intensity (see fig. 5 b). After subsequent cooling of the reaction chamber and formation of double stranded DNA, the fluorescence signal increased for positive PCR experiments (see fig. 5 c).
Example 4:
changes in fluorescence intensity following treatment of membranes with endpoint PCR solution from SYBR Green I assay
Here, the same experimental setup as described in example 3 was used, but only 1 membrane (PE-Sinter membrane from Polyan, pore size: 80-130 μm, thickness: 0, 6 mm; packing density of hydroxyl groups: 1, 3. mu. mol/cm)2) The membrane was divided into 2 compartments by an additional separation line made of polyvinyl chloride (PVC) dissolved in Tetrahydrofuran (THF).
The 2 solutions from the end-point PCR experiment of example 3 were pipetted into 2 separate compartments of the membrane. The film was again placed between 2 slides, clamped with a clamp, sealed with an adhesive foil and adjusted to room temperature or 80 ℃. Fluorescence intensity was measured in a 520nm channel by a LumiImager instrument (Roche applied Science, Mannheim, Germany). FIG. 6 shows images of such membranes with the corresponding fluorescence behavior during temperature cycling as outlined in example 3 (left compartment: positive PCR experiment, right compartment: NTC; a) room temperature, b)90 ℃, c)60 ℃, d) room temperature).
Example 5:
PCR reaction of beta-2 microglobulin in membranes by using probe-based detection means
PCR reaction mixture solutions of β -2 microglobulin were prepared using the Universal Probe library assay and in vitro RNA transcripts (from LightCyclerh-. beta.2M Housekeeping Gene Set, Roche Applied Science, Mannheim, Germany, Cat. No. 3146081) previously reverse transcribed using the Transc receptor first strand cDNA Synthesis kit (Roche Applied Science, Mannheim, Germany, Cat. No. 4379012) according to standard conditions described in the package insert. For theFinal PCR reaction to obtain about 10 of cDNA5The copies were mixed with primers (SEQ ID 3 and SEQ ID 4), Universal Probe Library probe (Probe Nr.42 for Universal Probe Library) and PCR Master mix (LightCycler TaqMan Master, Roche Applied Science, Cat. No. 04535286001). The final concentration of all PCR reagents in the PCR mixture was increased compared to the standard conditions from the package insert. The final concentrations were as follows: primer 1000nM, UPL probe 850nM, PCR mix 2, 1X.
This solution was pipetted onto a membrane (PE-Sinter membrane from Polyan, pore size: 80-130 μm, thickness: 0, 6 mm; packing density of hydroxyl groups: 1, 3. mu. mol/cm)2Dimension 17x 14mm) and subsequently sealed with a plastic foil (commercially available under the trade designation "depth folder" from Tropix Bedford, MA/USA under catalog number XF 030). The membrane was then placed between 2 slides, and the slides were clamped with clips, and the PCR reaction was performed under the following conditions: initial denaturation at 95 ℃ for 10 min and subsequent 45 amplification cycles, each with a denaturation step at 95 ℃ for 60 sec followed by an amplification step at 65 ℃ for 90 sec.
After the PCR reaction, the membrane was removed from the plastic foil and the liquid was obtained by centrifuging the membrane into a plastic lid. The resulting solution was pipetted onto a ready-to-use 4% agarose gel (Invitrogen, Carlsbad, CA/USA, catalogue number G501804) and compared to a reference PCR reaction performed with the same PCR reaction mixture on a LightCycler 2.0 instrument (Roche Applied Science, Mannheim, germany). On the LightCycler 2.0 instrument, the following PCR protocol was used: initial denaturation at 95 ℃ for 10 min and subsequent 45 amplification cycles, each with a denaturation step at 95 ℃ for 10 sec, followed by an amplification step at 65 ℃ for 30 sec and 72 ℃ for 1 sec, followed by a final cooling step at 40 ℃ for 30 sec. Figure 7 shows the corresponding gel, while the bands of 2 different membrane PCR reactions (indicated by II) yield the same amplicon length (quadruplicate indicated by I) as the reference PCR reaction.
Plus gel analysis, at the beginning of amplification (at cycle 1)) And at the end (cycle 45) the increase in membrane fluorescence intensity due to cleavage of the fluorophore of the UPL hydrolysis probe during PCR was measured by a LumiImager instrument (Roche applied science, Mannheim, germany) in the 520nm channel. As expected, in the 520nm channel, the signal intensity was 9, 8X 10 from the beginning for one membrane during the PCR reaction6Increased to 1, 1X 107And 9, 6X 10 from the beginning for another film6Increased to 1, 1X 107。