Matrix and method for purifying and/or isolating nucleic acids

The purification and isolation of nucleic acids from biological samples is a key technology in molecular diagnostics, epidemiology, food analytics, forensics and biological science. One of the most popular approaches involves binding of nucleic acids to silica surfaces in the presence of chaotropic agents. The principles of this approach are for example described by Boom et al (1990), J Clin Microbiol. 1990 March; 28(3): 495-503. Kits utilizing this technology are for example marketed by BioMerieux, Qiagen or Promega.

Nucleic acids dissolved ion a liquid sample have the ability to bind silica, i.e., amorphous SiO₂, in the presence of high concentrations of chaotropic salts (binding buffer). The latter denature biomolecules by disrupting the hydration shell surrounding them. This allows positively charged (e.g., sodium ions provided with the binding buffer) ions to form a salt bridge between the negatively charged silica and the negatively charged DNA backbone. In a next step, a low ionic strength buffer (low salt buffer) is being used to disrupt theses bindings by solubilizing the nucleic acids, in order to elute the nucleic acids.

Summary of the invention

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Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms a, an and the include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

The dependent claims are related to preferred embodiments. It is yet to be understood that value ranges delimited by numerical values are to be understood to include the said delimiting values.

According to the invention, a matrix material suitable for use in purifying and/or isolating nucleic acids from a biological sample is provided, which matrix comprises a surface comprising at least one element selected from the group consisting of Germanium, Tin and/or Lead; or at least one salt thereof.

Germanium (Gc), Tin (Sn) and Lead (Pb) belong to the Carbon Group in the periodic table, also called group 14 according to the new 1LPAC system. Compared to the remaining elements in the Carbon Group, i.e., Carbon (C) and Silicon (Si), the three former elements have in common a high density and atomic mass, plus a good electrical conductivity, which seperates them from Silicon and Carbon. Germanium (Ge), Tin (Sn) and Lead (Pb) thus form a subgroup with group 14. The followjng tables shows this clearly:

<td> element</td><td> atomic mass</td><td> densify (kg/m⁵)</td><td> electrical conductivity (S/m)</td>

<td> Carbon</td><td> 12,011</td><td> 2250-3510</td><td> 1 x 10 ⁴-3x 10⁶</td>

<td> Silicon</td><td> 28,086</td><td> 2330</td><td> 2,52 x 10 ⁴</td>

<td> Germanium</td><td> 72,59</td><td> 5323</td><td> 1,45</td>

<td> Tin</td><td> 118,71</td><td> 7310</td><td> 9,17 x 10⁶</td>

<td> Lead</td><td> 207,2</td><td> 11340</td><td> 4,81 x 10⁶</td>

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Furthermore, Germanium, Tin and Lead have a greater ionic diameter than Silicon.All these technical features contribute to significant differences in the binding reaction of Silicon, on the one hand side, and Germanium, Tin and Lead, on the other hand side, with the nucleic acid backbone.

In a preferred embodiment, said matrix comprises Germanium or a Germanium salt, preferably Germanium oxide.

Germanium a chemical element with the symbol Gc and atomic number 32. Germanium dioxide (GeO₂), also called Germanium Oxide (in contrast to Germanium monoxide, which is GeO) or Germania, is an inorganic compound, an oxide of Germanium. Its chemical formula is GeO₂. Other names include germanic acid, G-15, and ACC10380. It forms as a passivation layer on pure Germanium in contact with atmospheric oxygen. The forms of Germanium dioxide parallel, to an extent, those of silicon dioxide.

Hexagonal GeO₂ has the same structure as β-quartz (Germanium having coordination number 4); tetragonal GeO₂ (the mineral argutite) has the rutile-like structure of stishovite (Germanium having coordination number 6); and amorphous (glassy) GeO₂ is similar to fused silica. Germanium dioxide can be prepared in both crystalline and amorphous forms. Like Silica, it can be provided in a gel form, which is a granular, vitreous, highly porous form which, despite its name, is a solid having a large inner surface with pores in the nanometer range.

Because Germanium has a higher electronegativity than Silicon (2.02 vs 1.74), liquid-based deposition processes of, e.g., GeO₂on metal surfaces have a higher efficiency than with SiO₂. Further, due to that higher electronegativity the binding reaction between GeO₂ and nucleic acids is stronger, because the GeCh-domains have a higher polarity.

Generally, the matrix material can consist entirely of Germanium oxide. In a preferred embodiment, however, only the surface of the material comprising Germanium oxide, while the core areas of the material comprises other materials. Such embodiment can be used to add, to the nucleic acid binding capacity of Germanium oxide, other technical features which can

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WO 2013/021027 PCT/EP2012/065574 be useful in the present context. Further, this opens up the possibility to use cheaper materials than Germanium, or its derivatives, in the core areas.

In a preferred embodiment of the present invention, it is provided that said matrix material is provided in at least one shape selected from the group consisting of • Reaction, vessel coating • Particles • Powder • Fibres • Membrane

In case the matrix material is a membrane, such membrane can for example be used in a spin column, e.g., in. column-based nucleic acid purification. In case the matrix material is in form of particles, the latter can be used in particle-based nucleic acid purification systems. In case the matrix material is in form of a reaction vessel coating, nucleic acids can be bound the the walls of a reaction vessel for purification purposes. In case the matrix material is in form of fibres, a wool-like material can be produced which can be used in columns for nucleic acid purification. In case the matrix material is in form of powder, a suspension can be produced similar to glass milk, which has a greater surface area, and thus can bind more nucleic acids per unit volume than other regularly shaped silica matrices.

In another preferred embodiment of the present invention, it is provided that said particles have at least one feature selected from the group of • Spherical shape • Diameter between > 0,01 pm and < 100 pm

As used herein, the term spherical shape is not always required to be a true sphere or a nearly true sphere because the purpose is to compare it with such longitudinal shapes(like in fibres) or planar shapes (like in membranes). Such type of particles is also called beads, or nano- or microspheres.

Preferably, the mean diameter of the said partickes is in the range of > 0,05 pm and < 5 pm, even more preferred in the range of > 0,1 pm and < 1 pm. Particularly preferred, the mean diameter is in the range of > 0,15 pm and < 0,25 pm.

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In another preferred embodiment of the present invention, it is provided that said material is, at least in part, magnetically-responsive.

The term magnetically responsive material refers to any magnetic, paramagnetic or magnetizable material, The term also refers to the capacity of a material to migrate, relative to under the influence of a magnetic field.

In such embodiment, a magnet can be used to collect the matrix material, e.g., the beads, after they have bound the nucleic acids. In this embodiment, washing steps or elution steps are facilitated, particularly when Formalin Fixed Paraffin Embedded (FFPE) sample material is used (see below).

Preferably, the matrix material comprises, or consists, at least in part, of, an anorganic material.lt is particularly preferred that matrix material comprises a magnetic or paramagnetic material selected from the group consisting of • Iron oxide • Magnetic polymers • Gold

Iron oxide particles are for example commercially available as toner for photocopiers. These particles arc produced under very high standards and have thus a very even size distribution, arc chemically and have a high purity. Such type of particles, although witn a silkica coating, are for example marketed by Mobitec, Goettingen, DE. Alternatively, said iron oxide particles consist of hydrophilic Fe^Ch, which is for example available as BAYOXIDE E8706, E8707, E8709 and/or E8710. As regards the somehow surprising feature that Gold can have magnetic properties, reference is made to Trudel (2011), Unexpected magnetism in gold nanostructures: making gold even more attractive Gold Bulletin Volume 44, Number 1, 3-13.

In magnetic polymer beads, the particle matrix consists of either latex, polystyrene or silica with, e.g., homogeneously incorporated nanometer-sized iron oxide. Such type of beads is for example marketed as Dynabeads by life technologies.

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In a particularly preferred embodiment, magnetically responsive beads with, e.g., an iron oxide core and a Germanium dioxide coating are being used. According to another aspect of the invention, a method for purifying and/or isolating nucleic acids from a biological sample is provided, in which method a matrix material according to the invention is used.

In a preferred embodiment of said method, the nucleic acids to be purified and/or isolated are selected from the group consisting of DNA and or RNA. It is particularly preferred that the nucleic acids are genomic DNA mRNA and/or microRNA.

In a particularly preferred embodiment of said method, the biological sample is at least one selected from the group consisting of:

• Fresh tissue samples • Frozen tissue samples • Fixed tissue samples • Forensic or paléontologie samples, • Samples obtained from faeces, dried biological material, mummies, taxidermized organisms • Food samples, and/or • Plant samples

For fixed tissue samples, at least one fixative may used in a preferred embodiment which is selected from the group consisting of Neutral Buffered Formaline, Unbuffered Formaline, Glutaraldehyde, Ethanol, Acetone, Methanol, Methacam, Camoy's fixative, AFA-Fixative (Formaldehyde, Ethanol and acetic acid), Pen-Fix (alcoholic formalin fixative), Glyo-Fixx (glyoxal-based fixative), Hope (Hepes-glutamic acid buffer mediated organic solvent fixative), and/or Zinc Formal-Fixx (Formaldehyde fixative which contains zinc).

A preferred typuic of fixed tissue samples arc Formalin Fixed Paraffin Embedded (FFPE) tissue samples. Routinely, in tumor diagnosis tissue samples are taken as biopsies form a patient and undergo diagnostic procedures. For this purpose, the samples are fixed in formaline, embedded in paraffine and are then examined with immunohistochemistry methods. The formaline treatment leads to the inactivation of enzymes, as for example the

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WO 2013/021027 PCT/EP2012/065574 ubiquitous RNA-digesting enzymes (RNAses). For this reason, the mRNA status of the tissue (the so called transcriptome), remains unaffected.

However, molecular analysis in FFPE samples, particularly by means of nucleic acid amplification and detection, is a difficult manner because the fixation process crosslinks proteins and nucleic acids. Further, the process to dissolve nucleic acids from FFPE tissue which is usually done manually is highly error-prone. Another issue is that in FFPE samples, nucleic acids are often disrupted into very short fragments, which, although they are still long enough to be analyzed by PCR, pos problems when being isolated with standard means.

Such samples can successfully be treated with a preferred embodiment of the invention, in which magnetically responsive beads with, e.g., an iron oxide core and a Germanium dioxide coating are used. Because being magnetic the said beads can be used in an automatic environment, thus eliminating the errors caused by manual dissolving of nucleic acids from FFPE tissue. Further the beads can bind also small fragments of nucleic acids.

Regardless from the way the sample has been conserved, the sample type may comprise tissue sections, Tissue Micro Array cores, samples from needle aspirates, smear samples, microdissected samples, and samples obtained from cell culture

In another preferred embodiment of said method, the nucleic acids are purified and/or isolated in the presence of a chaotropic agent.

The term chaotropic agent as used herein refers to salts of particular ions which, when present in a sufficiently high concentration in an aqueous solution, cause proteins present therein to unfold and nucleic acids to loose secondary structure. It is thought that chaotropic ions have these effects because they disrupt hydrogen-bonding networks that exists in liquid water and thereby make denatured proteins and nucleic acids thermodynamically more stable than their correctly folded or structured counterparts.

In yet another preferred embodiment of said method, the purification and/or isolation comprises a step of focusing a magnetically responsive matrix material according to the invention by means of a magnetic field.

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In this embodiment, washing steps following the binding of nucleic acids are facilitated, because the matrix material with the nucleic acids can be immobilized temporarily, thus avoiding that they are washed away and thus get lost.

According to another aspect of the invention, a kit of parts suitable for use in a method according to the invention is provided, said kit comprising a chaotropic agent and, optionally, a matrix material according to the invention.

Preferably, said kit farther comprises a binding buffer and a low salt buffer.

As low salt buffer, TE buffer or water are preferably used. TE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or RNA. TE is derived from its components Tris, a common pH buffer, and EDTA, a molecule that chelates cations like Mg²\ A typical recipe for making 10:1 TE buffer is 10 mM Tris, (ad pH 8.0 with HCl) and 1 mM EDTA

The binding buffer comprises a chaotropic agent and a buffer, plus, optionally, adetergent and/or NaCl and/or KC1 can be added in high concentrations. In the latter case, the buffer is also called high salt buffer.

The Kit or method according to the invention preferably comprises at least one chaotropic agent selected from the group consisting of:

Urea

Thiourea

Guanidinium chloride

Guanidinium hydrochloride

Thiocyanates, like Guanidiniumthiocyanate

Perchlorates, like Lithium perchlorate or sodium perchlorate

Trichloracetates, like sodium trichloroacetate

Iodides, like sodium iodide

Barium salts

Urea is preferably used in a concentration of 6 · 8 mol/1. Thiourea is preferably used in a concentration of 2 mol/1. Guanidinium chloride is preferably used in a concentration of 6 mol/1. Lithium perchlorate is preferably used in a concentration of 4.5 mol/1

Preferably, said kit or method further comprises at least one agent selected from the group consisting of • Degrading enzyme • Detergent • Alcohol

Degrading enzymes include Proteases. Proteinase K is one of these, and actually works very well in these denaturing buffers; the more denatured the protein, the better Proteinase K works. Lysozyme, however, does not work in the denaturing and so lysozyme treatment is usually done before adding the denaturing salts. Detergents help with protein solubilization and lysis. Preferably, Triton™ X 100 is used as detergent. Alcohol is used to enhance and influence the binding of nucleic acids to the matrix, and for washing purposes. Preferably, ethanol and/orisopropanol are used

In a preferred embodiment, the kit according to the invention further comprises a magnetic separator. In this embodiment, washing steps following the binding of nucleic acids are facilitated, because the matrix material with the nucleic acids can be immobilized temporarily, thus avoiding that they are washed away and thus get lost Such magnetic separator can preferably be embodied in the form of a microtiter plate which can accommodate a number of micro reaction vessels, like Eppendorf™ tubes. Said separator may consist of a tablet, or a block, e.g., from Plexiglas™, with a number of wells (either for the samples themselves, or for accommodation of the Eppendorf™ tubes). In the lower section of the tablet, or block, one or more magnets (either permanent magnets, or electromagnets) are disposed, which attract the magnetically responsive matrix materials, e.g. the GeOz coated iron oxide beads.

Alternatively, said magnetic separator can consist of an individual tablet, or block, having the size of a micotiter plate, in which one or more magnets are disposed, which block can then be used together with a microtiter plate in a sandwich configuration. A standard size of such magnetic separator is 12,8 cm x 8,6 cm x 2,8 cm for use with standard 96 well microtiter plates.

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According to yet another aspect of the invention, the use of a kit, method or matrix according to the invention for at least one purpose selected from the group consisting of • Forensics • Molecular diagnostics • Food analytics, and/or • Plant analytics is provided

Figures and Experiments

Additional details, features, characteristics and advantages of the object of the invention are disclosed in the subclaims, and the following description of the respective figures and examples, which, in an exemplary fashion, show preferred embodiments of the present invention. However, these drawings should by no means be understood as to limit the scope of the invention.

Fig. 1 shows a schematic view of the method according to the invention.

Fig. 2 shows a magnetic separator as can be sued in the context of the present invention

Fig. 3 demonstrates the binding principle between GeO₂ coated surfaces and nucleic acids.

Examples

1. Production of a Matrix material according to the invention

NaaGeCh is produced by reaction of Sodium carbonate and Germanium dioxide when molten according to the following scheme

Na₂COj + GeO₂ -+ Na₂GeOj + CO₂

Anhydrous NazGeOj contains a chain polymeric anion composed of comer shared {GeOi} tetrahedral, and not a discrete GeCh² ion.

g of iron oxide particles as used for toner are given into 1000 ml of an aqueous 0.25% solution of NazGeOj. After stirring for an hour, the particles are filtered off, washed subsequently with water and ethanol, and are then dried. Alteematively, a 20 % solution of Na₂GeCh can be used.

Other ways to created GeO₂-coated matrix material comprise Plasma Enhanced Chemical Vapor Deposition and Chemical Vapor Deposition.

2. A nucleic acid purification kit according to the invention.

A non-limiting example of a nucleic acid purification kit according to the invention comprises at least the following items:

Binding buffer (500 μΐ): 5 M Guanidiniumisothiocyanate, 10 mM TrisHCI, 20 % Triton™, pH 8,8 Optionally, a NaCl and/or KC1 can be added in high concentrations

Washing buffer: 50 vol % ethanol, 20 mM NaCI, 10 mM Tris-HCl, pH 7,5

Low salt buffer (50 μΐ): 50 ul TE-buffer (10 mM Tris, 1 mM EDTA, pH 7,0)

GeOz coated particles (optional): 3 mg

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3. Purification of a ESRI nucleic acid with a matrix material according to the invention, and further amplification

RNA is isolated from formalin- fixed paraffin-embedded (FFPE) tumor tissue slice samples. The FFPE slicess are lysed and treated with Proteinase K for 2 hours 55°C with shaking. After adding a binding buffer (high salt + chaotropic salts) and GeOz-coated magnetic particles nucleic acids arc bound to the particles within 15 minutes at room temperature. On a magnetic stand the supernatant is taken away and beads are washed several times with washing buffer. After adding elution buffer (low salt) and incubating for 10 min at 70°C the supernatant is taken away on a magnetic stand without touching the beads.

After normal DNAse I treatment for 30 min at 37°C and inactivation of DNAse I the solution is used for reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR is run as standard kinetic one-step Reverse Transcriptase TaqMan(TM) polymerase chain reaction (RT-PCR) analysis on a ABI7900 (Applied Biosystems) PCR system for assessment of mRNA expression.

Raw data of the RT-PCR arc normalized to one a housekeeping gene according to standard methods.

Experiments shown that the determination of ESRI by RT PCR consistently yields better results than analysis by immunohistochemistry (IHC).