Method and kits of parts for the depletion and / or recovery of nucleic acids

Abstract

Methods and kits of parts for depleting and / or enriching nucleic acid comprising the use of a rare earth metal ion.

Description

[0001] Method and Kits of Parts For The Depletion and / or Recovery of Nucleic Acids

[0002] The present invention relates to methods and kits of parts for depleting and / or enriching nucleic acid.

[0003] It is now over 150 years since Friedrich Miescher first discovered nucleic acids (which he called nuclein, owing to its isolation from cell nuclei). The first extracts were derived from clinical material, but he soon adopted salmon sperm as a more convenient experimental system. Over the next 100 years, our understanding of the chemical properties of DNA, began to emerge as methods for the purification of DNA were developed. However, since this time, the methodology for the extraction and purification of DNA has remained largely unchanged. After cells containing DNA, which may be bacteria, yeast, plants or human tissues, are lysed with some form of chemical or physical breakage, the DNA is separated from proteins, metabolites (lipids, carbohydrates etc) using a combination of solvents, salts and more recently solid phase resins.

[0004] Perhaps the most significant change in the experimental procedures that have made the investigation of DNA routine, has been the introduction of standardised, commercial kits for DNA extraction and downstream analysis. DNA is most frequently produced as a template for DNA amplification via the polymerase chain reaction (PCR) and nucleotide sequencing. These kits provide scientists with ready to use solutions of reagents for the extraction, purification and concentration of DNA in both manual and automated formats. While cell extraction is usually tailored for the type of biological cell or tissue, the final stages of purification and concentration of DNA are generic: the DNA from archaebacteria is chemically the same as DNA from humans. Historically, the combination of traditional chemistry solvents, such as ethanol, phenol and chloroform etc, and simple salts, at high molar concentrations, have dominated practical nucleic acid research and manufacturing. However, while such methods have been central to the development of the field of molecular biology, the future translation of nucleic acid technology to the spheres of manufacturing is underway. Nucleic acids are now manufactured in kilogram quantities for vaccine production. In synthetic biology, nucleic acids are already central to diagnostics and are even proposed for new generations of computer technology.

[0005] The analysis of nucleic acids, including deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) is of major importance in biological, agricultural biotechnology (AgBio), medical and pharmacologic research, environmental research, forensics, ancestry testing, genotyping and diagnostics. For example, examination of the nucleotide sequence of the genomic nucleic acid of any given cell is sufficient to determine its biological origin and genetic identity: from one bacterial species to another and from one individual organism to another. In addition, genetic markers for the detection and / or prediction of diseases or phenotypic traits can be identified. Furthermore, analysis of RNA and DNA is also useful for identifying pathogenic bacteria, fungi and viruses. Thus, extracellular nucleic acids (so called 'cell-free DNA') are in particular useful in non-invasive diagnosis and prognosis and can be used e.g. as diagnostic markers in oncology, transplantation medicine or many other diseases.

[0006] However, most of these methods for analysing nucleic acids require nucleic acids as templates or substrates, which are essentially isolated and purified away from most other cellular components and further contaminants within a sample in preceding steps, for example during or immediately after a cell lysis step. In other cases, the nucleic acid may be regarded as a contaminant. For example, in the production of recombinant proteins using host cells such as bacteria, the cells are isolated then lysed at the end of the fermentation process. It may be desirable to remove nucleic acids from the lysate prior to the purification of the target protein product.

[0007] Various methods are known for the isolation of nucleic acids like DNA and RNA from aqueous samples comprising said nucleic acids, e. g. biological samples lysed in an aqueous lysis buffer.

[0008] In the field of molecular diagnostics increasing numbers of samples lead to bottlenecks in workflows, as either the assays for nucleic acid analysis or the isolation of nucleic acids to achieve sufficient purity for analysis are either too slow or too cost-intensive, or both.

[0009] Purification of nucleic acids using known methodology is often slow because the reagents used to lyse the sample and bind the nucleic acid are slow acting and must be washed out and removed prior to downstream analysis because they inhibit the downstream assays. Typically, nucleic acid purification with solid supports is done in the sequence of lysis, binding, two to three washing steps, drying and elution. This usually results in kits that use a lot of plastic in processing, generate large amounts of environmentally harmful waste as the reagents are not often recyclable and are expensive due to the large amount of the types of chemicals needed for purification. In addition, each step increases the time from sample to result.

[0010] Known methods commonly used for the purification or removal of nucleic acids from biological and / or biochemical samples include precipitation at low temperatures (typically between -20 to 4°C) following the addition of a combination of low molecular weight alcohols (including ethanol and isopropanol), in particular ethanol, and monovalent cations at high concentrations (including potassium, sodium, lithium or ammonium). The operating concentrations of ethanol (for example) are typically greater than 8M and the neutralising cation above 300mM, final concentration. The modulation of pH during precipitation has been used to separate plasmid (the alkaline lysis method) from genomic DNA [Sambrook, J., Fritsch, E. R., & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press] (incorporated herein by reference). In addition, cationic polymers such as polyethylene imine have been used to aid in the purification of DNA binding proteins, for many years [See Atkinson A, Jack GW. Precipitation of nucleic acids with polyethyleneimine and the chromatography of nucleic acids and proteins on immobilised polyethyleneimine. Biochim Biophys Acta. 197321;308(7):41-52. doi: 10.1016 / 0005-2787(73)90120-2] (incorporated herein by reference). Other non-selective reagents such as streptomycin sulfate have also been incorporated in DNA depletion protocols [Oxenburgh MS, Snoswell AM. Use of streptomycin in the separation of nucleic acids from protein in a bacterial extract. Nature. 1965 Sep 25;207(5004):1416-7. doi: 10.1038 / 2071416a0] (incorporated herein by reference). E and H Hammarsten first describe lanthanides binding nuleic acid (Hammarsten, E. " The biological significance of nucleic acid compounds." Biochem. Z 144 (1924): 383-466. Original Title in German: Hammarsten, Einar. Zur kenntnis der biologischen bedeutung der Nucleinsaureverbindungen. Diss. J. Springer, 1924.) which was subsequently cited by Chargaff in 1948 (Chargaff, Erwin, et al. " The composition of the desoxypentose nucleic acids of thymus and spleen." Journal of Biological Chemistry 177.1 (1949): 405-416) and Stern in 1953 (STERN KG, STEINBERG MA. Desoxyribonucleic acid complexes of rare earths. Biochim Biophys Acta. 1953 Aug;11(4):553-8). The methodologies relate principally to chromatin but significantly, the chemical risk to the deoxyribonucleic acid and the potential health risks were deemed significant. Furthermore, function of the DNA was perceived to have been greatly compromised.

[0011] Early attempts to develop protocols for the purification of nucleic acids combined the use of solvents (mainly to remove proteins) and aqueous salt solutions, often accompanied by the use of organic solids such as diatomaceous earths and cellulose fibres, acting as co-precipitants and "filterable" solid phases. The use of contemporary chromatography resins with batch-to-batch consistency as part of the purification of nucleic acids was introduced commercially by Qiagen in the 1980s, (see also US-5808041-A - Nucleic Acid Purification Using Silica Gel and Glass Particles, assigned to Promega) (incorporated herein by reference) followed by the development of magnetic bead technologies [Safarik I, Ptackova L, Safarikova M. Large-scale separation of magnetic bio affinity adsorbents. Biotechnol Lett. 2001;23:1953-1956], Rapid analysis of protein-nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography [Dickman MJ, Sedelnikova SE, Rafferty JB, Hornby DP. Rapid analysis of protein-nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography. J Biochem Biophys Methods. 2004 Jan 30;58(1):39-48.] (incorporated herein by reference) and the introduction of extraction devices from spin columns to vacuum manifolds, and today, automation via robotic platforms has become commonplace in molecular diagnostic laboratories worldwide [Li Y, Liu S, Wang Y, Wang Y, Li S, He N, Deng Y, Chen Z. Research on a Magnetic Separation-Based Rapid Nucleic Acid Extraction System and Its Detection Applications. Biosensors (Basel). 2023 Sep 23;13(10):903. doi: 10.3390 / bios13100903] (incorporated herein by reference).

[0012] Furthermore, the addition of nuclease enzymes, such as deoxyribonuclease (DNase) or ribonuclease (RNase) from (for example) pancreatic tissues have been widely used to remove one or both classes of nucleic acids from cell extracts or experimental samples. However, where downstream processing of the nucleic acids requires the maintenance of their biochemical integrity, such as the use of the nucleic acid as a template for enzymatic amplifications (e.g. the polymerase chain reaction, or isothermal amplification) the addition of nucleases is considered inappropriate [Sambrook, J., Fritsch, E. R., & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press] (incorporated herein by reference). Specialist applications continue to be developed for the depletion and / or enrichment of nucleic acids in diagnostic testing. For example, specific chromatographic resins may be combined with non-ionic detergents and an anionic ion-pairing reagent to form a solid phase platform for depleting nucleic acids from cell extracts (eg Sbeadex from LGC (WO 2024 / 160728 Al: Method for isolating and / or purifying nucleic acids)). Similarly, the direct coating of chromatographic, magnetic beads, with a polyionic polymer, may be used to enrich nucleic acids in the liquid phase (including circulating free DNA or cfDNA), which shows promise for the analysis of "liquid biopsies" (eg Biocaptiva's msX™ technology, US12, 037, 578 ).

[0013] Accordingly, speedier methods for the isolation and / or purification of nucleic acids are desirable, which allow a quick and reliable isolation and / or purification of the nucleic acids, preferably in an environmentally friendly manner.

[0014] Advantageously, the present invention overcomes or alleviates one or more of the problems associated with the prior art.

[0015] Definition of terms

[0016] As used herein, the term "rare earth element" refers to any one of the following elements:

[0017] Scandium, Yttrium, Lanthanum, Cerium, Praseodymium, Neodymium, Samarium, Europium, Gadolinium, Terbium, Dysprosium, Holmium, Erbium, Thulium, Ytterbium or Lutetium. As used herein, the term "nucleic acid" refers to an organic polymer found in living cells, especially DNA or RNA, whose molecules consist of many nucleotides linked via a phosphodiester bond. Nucleic acids may be double-stranded, wherein two polymer chains form a non-covalent duplex, sustained primarily by hydrogen bonding and "base-stacking" (hydrophobic) interactions. Nucleic acids may also be single stranded, or comprise regions of single strandedness, interspersed with double-stranded regions formed by inter- and / or intramolecular Watson and Crick base pairs.

[0018] As used herein, the term "nucleic acid precipitate" refers to nucleic acids that, by virtue of their interaction with certain compounds (herein rare earth salts) are rendered insoluble in aqueous solutions.

[0019] As used herein, the term " Ksp" refers to the solubility product is a constant that defines the mole fraction of a sparingly soluble salt, such as a rare earth phosphate, at a given temperature and pressure in water (or a defined solvent).

[0020] As used herein, the term "magnetic moment" (p) refers to the magnetic strength and orientation of a magnet that generates a magnetic field and it defines the torque experienced by a magnet in an externally applied magnetic field (usually expressed in units of Bohr Magnetons, PB).

[0021] As used herein, the term "resin" refers to the name given to a chromatography matrix, especially one used routinely in the purification of biological molecules, including, but not limited to, nucleic acids, proteins, carbohydrates, lipids and metabolites. Examples of resins include agarose microbeads and related commercial versions of such beads, which may in-turn be chemically modified to incorporate metal ions or cationic and anionic polymers.

[0022] As used herein, the term "polymerase chain reaction" refers to the method by which low concentrations of nucleic acids (referred to as templates) can be amplified in the presence of a thermostable DNA polymerase, nucleotides (often referred to as dNTPs or NTPs), together with two or more, sequence-specific DNA primers (oligonucleotides typically between 20 and 50 nucleotides in length) and detected using some form of spectral, fluorometric or radioactive technology.

[0023] As used herein, the term "lysis" refers to the process by which living cells and / or tissues are disrupted either by mechanical, chemical, thermal or enzymatic means, in a vessel that captures the soluble and insoluble components of the cell. Following centrifugation, the soluble and insoluble components of the lysed cell can be separated and recovered separately.

[0024] As used herein, the term "agarose gel electrophoresis" refers to a method by which nucleic acids can be separated on the basis of size (conveniently between 100 to 40000 nucleotides) in an agarose, or polyacrylamide gel following the application of a voltage for around 30-60 minutes. The results of the separation are typically visualised by the addition of a nucleic acid specific, fluorescent dye (eg ethidium bromide or SYBR green), to the gel, followed by photography of the gel illuminated with ultraviolet light, or light of wavelength that leads to the excitation of fluorescence during the application of those method and reagents.

[0025] As used herein, the term "chelation" and "chelator" refers to the formation of a water-soluble complex between a metal ion and a complexing agent. It usually does not dissociate easily in solution but forms an inert complex. In this case, chelators such as EDTA and EGTA, form soluble complexes with rare earth metal ions.

[0026] The present disclosure may be embodied in various different forms and should not be construed as being limited to only the illustrated embodiments herein. Rather, these embodiments are provided as examples so that this disclosure will be thorough and complete, and will fully convey the aspects and features of the present disclosure to those skilled in the art.

[0027] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Further, the use of "may" when describing embodiments of the present disclosure refers to "one or more embodiments of the present disclosure." In the following description of embodiments of the present disclosure, the terms of a singular form may include plural forms unless the context clearly indicates otherwise.

[0028] As used herein, the term "substantially," "about," and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent deviations in measured or calculated values that would be recognized by those of ordinary skill in the art. Further, if the term "substantially" or "about" is used in combination with a feature that could be expressed using a numeric value, the term "substantially" or "about" denotes a range of + / - 5% of the value centered on the value or retains most, if not all, of the function.

[0029] It will be further understood that the terms "include," "comprise," "including," or "comprising" specify a property, a region, a fixed number, a step, a process, an element, a component, and a combination thereof but do not exclude other properties, regions, fixed numbers, steps, processes, elements, components, and combinations thereof.

[0030] The term "solution" as used herein in particular refers to a liquid composition, preferably an aqueous composition. It may be a homogenous mixture of only one phase but it is also within the scope of the present invention that a solution comprises solid additives such as e. g. precipitates, in particular of contained chemicals such as stabilizing agents.

[0031] The term "biochemical activity" is used to indicate the properties of material prepared using the methods described herein, that retains the in vitro chemical properties, by composition, structure and activity (or function) of those found in living cells (in vivo).

[0032] The term "biological activity" is used to indicate that any material prepared by the methods described herein retain their biochemical activity (ies) in a living cell or whole organism, with no detriment to the normal physiological behaviours of that cell or organism. Summary of the Invention

[0033] In accordance with a first aspect of the present invention there is provided a method for depleting and / or enriching nucleic acid comprising the steps:

[0034] (a) contacting a nucleic acid containing sample with a rare earth metal ion (including lanthanoid ions) having a metal phosphate Ksp of less than 10-24to form a metal nucleic acid precipitate and a supernatant;

[0035] (b) separating the precipitate from the supernatant; and

[0036] (c) collecting the precipitate to thereby obtain the nucleic acid complexed with the rare earth metal ion.

[0037] The metal ions of the present invention may be derived from one or more rare earth metals. This includes Scandium (Sc), Yttrium (Y) and the lanthanoids. The lanthanoid may comprise any one or more of the following: Lanthanum (La), Cerium (Ce), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), and Lutetium (Lu). The present invention has been demonstrated using the 16 non-radioactive rare earth metals (and omitted Promethium) on the basis that these are likely to have greater practical utility.

[0038] Table 1 Structural and electronic properties (from Beaudry and Gschneidner 1978, Handbook on the Physics and Chemistry of Rare Earths, Chapter 2) Electeoa Radins / a Crystal Lattice Parameter.

[0039] Element Z A Coalf Ionic Metallic Structure <: pm <■ pm e / a Scaadfaia Se a, 45 'Af-p - Ml hep 3M9 ®8.8 L59B Ytrium Y.<» tr U w 180.1 hep 364.8 5® J 1571 Lanftanuo Lo 57 139 4 / • 526s *:106.1 1W dhcp 3774 12171 8® Cerium W 4f‘ < A.?6s ’ 103.4 1825 fee 516.1 - - Pra edyiniutn Pr. O 141 Gi'Ko 181.8 dliep 367.2 1188,3 OS1 Neodymium Nd ® 144 4f' CWfe -' ®,5 Mil dhcp W& S 11795 3.® Pr.mietlnum Pm 61 145 4. C >‘5 J® ■ ' 97,9 181.1 dhcp 865 11® 3,19 Samariwa Sm 4< 9S,4 180,4 rliem 3®S *7J£gi Europium Eu K> Ixi 4 / ' < 526s •" 95.0 20<2 bee 458.3 — — Gadeimmm Gd 64 137 4’“’ G2A; »,8 180.1 hep 368.4 578.1 1591 Terbium Tb ® 1.39 4; ' 178.3 hep 360.6 569.7 1580 Dy pro.ium Dy 66 1® 4; ’ \3rfKs •' 80S 177.4 hep 350 565.0 1.573 Holmium Ho 67 163 1W6 l»ep 357.1 ®1.8 15W ErbiWt Er 61 167 1BJ ®5S «®,5 15® Thulium Im ® im 4J< N®® M9 174.6 hep ®. S 555,4 1570 Itwbiuia Yb A 178 85JB 193S fee 54&5 - -

[0040]

[0041] Lutetium Lu 71 175 4J’< 526s - 84.8 173.5 hep 350.5 »S 1583

[0042] Advantageously, the present invention may provide the precipitation of nucleic acid using rare earth salts, and their recovery using a simple chelating agent at physiological pH (such as EDTA). Unlike all other nucleic acid precipitation agents, the working concentration of the key rare earth trivalent metal ion component is typically in the micromolar range (compared with ethanol and monovalent cations of >10M and >250mM respectively).

[0043] The nucleic acid that is depleted and / or enriched according to the present invention may retain at least substantially its function. Advantageously, the nucleic acid retains its function. The function of the nucleic acid refers to its biochemical and / or biological activity.

[0044] Advantageously, the rare earth metal ion may be present in step a) at a concentration of less than lOmM. More advantageously, the rare earth metal ion may be present in step a) at a concentration of about lOmM or less, the rare earth metal ion may be present in step a) at a concentration of about ImM or less, rare earth metal ion may be present in step a) at a concentration of about 10 pm to about ImM, the rare earth metal ion may be present in step a) at a concentration of about 25 pm to about 100 pm, the rare earth metal ion may be present in step a) at a concentration of about 40 pm to about ImM, the rare earth metal ion may be present in step a) at a concentration of about 40 pm to about 250 pm, or the rare earth metal may be present in step a) at a concentration of about 50 pm.

[0045] Advantageously, the rare earth metal ion may be present in step a) at a concentration of less than lOmM. More advantageously, the rare earth metal ion may be present in step a) at a concentration of lOmM or less, the rare earth metal ion may be present in step a) at a concentration of ImM or less, rare earth metal ion may be present in step a) at a concentration of 10 pm to about ImM, the rare earth metal ion may be present in step a) at a concentration of 25 pm to 100 pm, the rare earth metal ion may be present in step a) at a concentration of 40 pm to ImM, the rare earth metal ion may be present in step a) at a concentration of 40 pm to 250 pm, or the rare earth metal may be present in step a) at a concentration of 50 pm. Advantageously, the concentration step may be rapid (seconds), occurs at room temperature, is fully reversible by the addition of one or more molecular chelators (including EDTA at a concentration of <lmM) and importantly has no negative impact on the biochemical and biological, downstream use of the purified and concentrated nucleic acid.

[0046] Examples of applications of the present invention include but are not limited to:

[0047] 1. The enrichment of nucleic acids from dilute samples for downstream diagnostic analysis 2. The depletion of nucleic acids from proteins for research and manufacturing purposes 3. Preparation of genomic DNA for forensic, ancestry, environmental or sterility analysis. 4. Preparation of cell-free DNA (cfDNA) for diagnostics including liquid biopsy

[0048] 5. Preparation of plasmid DNA for gene expression

[0049] 6. Preparation of PCR products for Next Generation Sequencing

[0050] 7. Preparation of cDNA libraries

[0051] 8. Purification and concentration of DNA or RNA oligonucleotides

[0052] 9. Purification and concentration of DNA and RNA oligonucleotides

[0053] There are many possible applications for nucleic acid isolation, but they can be divided into two main categories. Where the nucleic acid is a contaminant that needs to be removed: this will be referred to as "depletion". Where the purification and concentration of nucleic acid is the aim: this will be referred to as "enrichment". All biological nucleic acids (DNA, RNA and those containing modified bases, such as N-6-adenine, C5-methylcytosine etc), are characterised by the presence of phosphodiester backbone.

[0054] Applicants have surprisingly shown that soluble rare earth salts may be used for the selective and highly efficient depletion of nucleic acids from purified, partially purified nucleic acid materials, chemically, or enzymatically generated nucleic acid-containing materials, including nucleic acids in complex biological extracts. Applicants have also surprisingly shown that biochemical and biological activity of the nucleic acid is retained. Lanthanide ions have been associated with scission of DNA molecules [Ciesiotka J, Marciniec T, Krzyzosiak W. Probing the environment of lanthanide binding sites in yeast tRNA(Phe) by specific metal-ion-promoted cleavages. Eur J Biochem. 1989 Jun 15;182(2):445-50] so the retention of such activity is surprising.

[0055] The precipitation process can be from a solution containing the dissolved trivalent metal or the precipitation process can be onto an ion exchanger comprising the trivalent metal ion. In some embodiments of the invention, the ion exchanger comprises a bead or beads. In some embodiments of the invention, the ion exchanger is packed into a column bed, and in others the bead may be a suitable "coated" magnetic micro-bead or particle.

[0056] After precipitation, the insoluble rare earth-nucleic acid complex may be concentrated into a pellet. The precipitated complex can be recovered by removing the trivalent metal ion either by sequestering the metal ion with a chelating agent, by electrochemical means using the processes of electrophoresis or electrolysis, or by the process of metathesis, in which the insoluble rare earth-nucleic acid complex is mixed with a molar excess of a soluble phosphate salt in solution (such as, but not limited to potassium phosphate). The metal ion may also be removed by lowering the pH, although this is known to promote hydrolysis of the nucleic acid and is therefore not suitable for downstream applications. The recovered nucleic acids retain their biological / biochemical activity.

[0057] The rare earths are characteristically trivalent (although quadrivalent and divalent forms exist) and salts of the lanthanoids may be highly soluble with the nitrate and chloride salts capable of attaining concentrations in aqueous solutions of around 4M at room temperature. By way of complete contrast, the phosphate salts of lanthanum (La) and cerium (Ce), are amongst the most insoluble compounds on Earth.

[0058] The addition of rare earth ions to solutions containing nucleic acid leads to the spontaneous formation of a highly insoluble precipitate, which in one embodiment, can be readily isolated by removing the aqueous phase by centrifugation. The precipitated, rare earth-nucleic acid complex may be readily re-solubilised by the addition of a chelating agent (including EDTA and tricarboxylic acids, including citric acid) in 20-fold molar excess. The re-dissolved nucleic acid is completely biologically active in respect of biological and enzymatic processing and is non-toxic.

[0059] The isolation and / or purification method according to the present invention has remarkable advantages over the methods used in the prior art in that it is rapid, may be conducted at room temperature, may provide a high purity of extracellular nucleic acids, has reagents that are recyclable and may not affect bioactivity.

[0060] Nucleic acid(s) which can be depleted and / or enriched using the method of the present invention include DNA and RNA, in particular genomic DNA (gDNA), plasmid DNA, PCR-fragments, cDNA, rRNA, mRNA, miRNA, cfDNA, siRNA as well as oligonucleotides and modified nucleic acids such as so-called peptide or locked nucleic acids, respectively, (PNA or LNA), of microbial, including viral, bacterial and fungi, human, animal or plant origin. In addition, also hybrids formed of DNA and RNA can be purified, without being limited to these.

[0061] The sample to be processed by the method of the present invention may represent any sample comprising nucleic acids, and preferably is a biological sample, either in its natural state or in a processed form. Preferably the samples may include body fluids such as blood, serum, sputum, faeces, plasma, sperm, cerebrospinal fluids, saliva, etc., human, animal or plant tissues and tissue cultures, microbial, human, animal or plant cells and cell cultures, and human or animal organs or parts thereof, such as for example liver, kidney or lung. In addition, the samples may represent fluid samples such as waste or drinking water, juices, or food without being limited to these. In addition, the sample may represent a processed biologic sample such as for example a human, animal or plant cell lysate, bacterial lysate, aqueous or buffered solutions of a sample, or gels. If the nucleic acids to be isolated are present in a cellular material, it may be preferred to first destroy the cellular material according to any method known from the state of the art for releasing the nucleic acids from a cell, such as for example by mechanical, chemical or enzymatic lysis, before further processing them according to the method of the present invention. Preferably the cellular material is lysed to release the nucleic acids and known lysis buffers and methods can be used, such as contacting with ionic and non-ionic detergents, hypotonic solutions of salts, protease, chaotropic agents or solvents, grinding tissue or adding other lytic enzymes forming a nucleic acid containing sample, also referred herein as "lysate". The lysate is preferably a lysis solution of organic material as sample and lysis buffer.

[0062] Examples of lysis methods include sonication, boiling, homogenization, treatment with detergents or treatment with lysozyme or proteinases (e.g. Proteinase K). Examples of lysis buffers are nonionic detergents such as Triton X-100 which are often used in the presence of chaotropic agents such as guanidine hydrochloride, guanidine isothiocyanate and acetonitrile to produce a nucleic acid containing solution or lysate.

[0063] The precipitating agent or the chelating agent may be associated with a solid phase. Typically, the rare earth salt would be used to charge a strong cation exchange resin, such as SP Sepharose, or alternatively coupled to an agarose bead, coated with a chelating agent such as nitriloacetic acid (NTA). The immobilized rare earth ions would then capture soluble nucleic acid from a sample, and elution may be achieved by the addition of an excess of chelating agent. In some applications, rare earth ions may be removed from a suspension of nucleic acid, in a solid phase extraction process

[0064] The solid phase used in the method of the present invention may be present as a filter in a spin column but preferably may be magnetic, including paramagnetic, ferrimagnetic, ferromagnetic or superparamagnetic materials. The solid phase of the present invention is not particularly limited to a special form and may be in the form of for example particles, including magnetic particles, beads, including magnetic beads. Most preferably the particles may be paramagnetic beads or particles.

[0065] In step (b) the particles with the bound nucleic acids are separated from the remaining binding mixture. Thereby, the particles with the bound nucleic acids are collected. For this purpose, any means known in the art can be used. Suitable means include but are not limited to magnetic separation, centrifugation, sedimentation, the application of a vacuum, filtration and the like.

[0066] The lanthanoids are also known as f-block elements because their electronic structure involves the gradual filling of the f electron shell. An f shell has 7 orbitals and these orbitals are incrementally filled up across the series of lanthanoids with increasing atomic (and electron) number as shown in the table below (Lanthanide and Actinide Chemistry and Spectroscopy

[0067] Editor(s): Norman M. Edelstein Volume 131, September 23, 1980 Copyright © 1980 AMERICAN CHEMICAL SOCIETY) Table 2 magnetic moments of the tripositive aqueous ions of the lanthanoids and lutetium

[0068] ion Electronic configuration p / pB

[0069]

[0070]

[0071] La3*

[0072]

[0073] diamagnetic Ce3* if12.51

[0074] Pr3* 4f23.53

[0075] Nd3* 4f® 3.55

[0076] Pm3* 4f* 2.68

[0077] Sm3* 451.46

[0078] F.i ’ 4f 1

[0079] Gti3* 4fr8.00

[0080] Tb3* 9.33

[0081] Dy3* 4f® 10.55

[0082] Ho3* 4f1® 10.40

[0083] Er3* 4f« 0.50 Tm®* 4f127.35

[0084] 4.30

[0085]

[0086] ciiamagnefc

[0087] Some of the lanthanoids are inherently paramagnetic. The lanthanoid ions that show the greatest paramagnetic effect are Dysprosium (Dy) and Holmium (Ho) which have the largest number of unpaired electrons in their f shell (the maximum being 7 - one unpaired electron in each of the 7 orbitals in the f shell). Lanthanum and Lutetium are diamagnetic because they have no unpaired electrons. In an embodiment, magnetic beads may also be employed for step c). Owing to the inherently paramagnetic character of certain lanthanoids, the removal of the precipitate may be performed using a magnet with or without the use of beads. Advantageously, an electromagnet is employed so that the precipitate would fall off the magnet once the electromagnet is turned off. In such a method, a lanthanoid with a large magnetic moment (such as Dysprosium) would be the preferred nucleic acid binding lanthanoid.

[0088] The method of the present invention may be carried out at a temperature greater than or equal to 4°C. Advantageously the method is carried out at a temperature between 4 and 20 °C. More advantageously, the temperature is 4 °C.

[0089] In accordance with a further aspect of the present invention, there is provided a kit of parts to perform the method described hereinabove, comprising:

[0090] i) one or more rare earth metal ion having a valence of at least +3 and a metal phosphate Ksp of less than IO-24; and

[0091] ii) optionally one or more wash solutions and

[0092] iii) one or more elution solutions comprising a chelating agent.

[0093] The kit can be used in order to perform the method according to the first aspect. The advantages were described above. The isolated nucleic acids are of high quality and purity. The method thereby becomes more reliable which is an important advantage in particular when extracellular nucleic acids are isolated for the AgBio, medical and / or diagnostic field. The kit may comprise the binding solution b). Details regarding the binding solution were described in detail above and it is referred to the above disclosure which also applies here. As discussed above the binding solution according to the invention may comprise i) at least one nonionic detergent in a concentration which is selected from 5% (w / v) to 70% (w / v), 8% (w / v) to 40% (w / v), 10% (w / v) to 30% (w / v) and 20% (w / v) to 25% (w / v).

[0094] The rare earth metal ion may be provided at a concentration of less than lOmM. More advantageously, the rare earth metal ion may be provided at a concentration of about lOmM or less, about ImM or less, about 25 pm to about ImM, about 25 pm to about 100 pm, about 40 pm to about ImM, about 40 pm to about 250 pm, or about 50 pm.

[0095] The present invention will now be illustrated but not limited by reference to the following Figures and Examples wherein:

[0096] Fig. 1 shows agarose gel electrophoresis of a 10-0.5kbp DNA ladder (New England Biolabs or NEB) in the presence and absence of La(N03)3;

[0097] Fig. 2 shows the detailed analysis of the concentration dependence of DNA precipitation by La(N03)3;

[0098] Fig. 3a shows the visualisation of the precipitation of DNA by LaCI3and recovery of the precipitated DNA with ImMEDTA in the presence of SYBR Green; Fig. 3b is a schematic diagram showing a method for the enrichment and depletion of soluble nucleic acids in accordance with the present invention;

[0099] Fig. 4 shows the demonstration of the biochemical integrity of DNA precipitated by LaCIs, recovered with EDTA (and EGTA) and shown to be a substrate for restriction endonucleases (4A), T4 DNA Ligase (4B) and PCR directed amplification (4C and 4D);

[0100] Fig. 5a shows the demonstration that RNA can be precipitated from cell extracts using LaCIs; Fig. 5b shows a schematic diagram of a method of enrichment and depletion of cellular nucleic acids in accordance with the present invention;

[0101] Fig. 6a shows La3+ions can be immobilised on a strong cation exchanger (SP Sepharose) and used to capture DNA from solution, and the DNA subsequently released by the addition of a chelating agent (EDTA);

[0102] Fig. 6 b shows a schematic diagram of a method of enrichment and depletion of cell-free DNA using rare earth ions immobilized on magnetic beads in accordance with the present invention, and

[0103] Fig. 7 shows the demonstration of the biochemical activity / integrity of RNA isolated by precipitation with LaCIs followed by recovery with 1mM EDTA. The RNA was used as a substrate for a quantitative RTPCR experiment and the product yield and amplification profiles were shown to be dependent on the presence and relative concentration of the RNA template.

[0104] Some of the data supporting the claims will take the form of photographs taken of agarose gel electrophoresis experiments, imaged using fluorescent dye detection. As a result, the images, when converted to monochrome versions, show DNA as a white "band" on a black background. In

[0105] some cases (annotated accordingly), the images have been inverted to bring out the salient

[0106] features (here the nucleic acids appear as dark bands on a white background, equivalent to

[0107] photographic negatives).

[0108] Figure 1

[0109] Figure 1 shows agarose gel electrophoresis of a 10-0.5kbp DNA ladder (NEB) in the presence and

[0110] absence of LafNOsh. 250ng of a DNA ladder was mixed with a range of concentrations of LafNOsh

[0111] in a total volume of 20pl. The sample was centrifuged at 10 OOOrpm for 2 minutes and the

[0112] supernatant applied to the gel and electrophoresed for 40 minutes. Nucleic acid was detected via

[0113] fluorescence using Gel green. This figure demonstrates the precipitation of DNA by LafNOsh.

[0114] Lane Sample

[0115] 1 DNA molecular weight ladder (10000 (top band) to 200bp) (NEB)

[0116] 2 250ng DNA (lane 1) in distilled water

[0117] 3 250ng DNA ladder in 50mM La(N03)3

[0118] 4 250ng DNA ladder in 5mM La(N03)3

[0119] 5 250ng DNA ladder in 0.5mM La(N03)3

[0120] 6 250ng DNA ladder in 0.05mM La(N03)3

[0121] 7 250ng DNA ladder in 0.005mM La(N03)3

[0122] 8 250ng DNA ladder in 0.0005mM La(N03)3

[0123] 9 250ng DNA ladder in 0.00005mM La(N03)3

[0124]

[0125] Figure 2.

[0126] Lane Sample

[0127] 1 lOOng Bacteriophage lambda DNA (NEB) lOpM La(N03)3

[0128] 2 lOOng Bacteriophage lambda DNA (NEB) 20pM La(N03)3

[0129] 3 lOOng Bacteriophage lambda DNA (NEB) 30pM La(N03)3

[0130] 4 lOOng Bacteriophage lambda DNA (NEB) 40pM La(N03)3

[0131] 10 5 lOOng Bacteriophage lambda DNA (NEB) 50pM La(N03)3

[0132]

[0133] Detailed analysis of the concentration dependence of DNA precipitation by LafNChh. lOOng of bacteriophage Lambda DNA (NEB), in a total volume of 20pl was mixed with increasing concentrations of LafNChh. The samples were immediately centrifuged, and the supernatants analysed by agarose gel electrophoresis, as in Fig.l. Relative intensities of the fluorescent DNA species are plotted below the DNA species visualised on the agarose gel. At a final concentration of 50pM LafNChh, no DNA is detected on the gel, consistent with its precipitation at this concentration. Figure 3a

[0134] Tube Sample

[0135] 1 500ng Phage lambda DNA (NEB) in 10ml water / SYBR Green

[0136] 2 500ng Phage lambda DNA (NEB) in 10ml water / SYBR Green + 50pM LaCI3

[0137] 3 Pellet obtained after centrifugation (of sample 2)

[0138] 4 Supernatant obtained after centrifugation (of sample 2)

[0139] 5 Addition of a solution of 50pl (lOmM) Tris / (lmM) EDTA, pH 7.5, leads to recovery of soluble

[0140] DNA (as revealed by SYBR-green fluorescence)

[0141]

[0142] Chelation-mediated recovery of 500ng bacteriophage lambda DNA, dissolved in 10ml distilled water, precipitated by the addition of LaCIs (to a final concentration of 50pM), followed by centrifugation. The pellet was dissolved in 50pl 10mMTris-CI containing ImM EDTA.

[0143] Fig. 3b shows a schematic diagram of a method of enrichment and depletion of soluble nucleic acids according to the invention.

[0144] Step 1: Soluble nucleic acids 10 are contacted with a solution of (or powdered) rare earth salt to yield a final concentration of, for example, 0.1-10mM LaCI3 to form a metal nucleic acid precipitate and a supernatant 12;

[0145] Step 2: the tube is vortexed and the precipitate is recovered by centrifugation thus the metal nucleic acid precipitate 14 of insoluble nucleic acids is enriched;

[0146] Step 3: a small volume of chelating agent is added (depending on the next protocol) to the nucleic acid pellet and the mixture vortexed. The solution 16 is now ready for downstream applications; Step 4: the supernatant is collected to give a nucleic-acid free solution 18. Figure 4

[0147] This figure demonstrates the biochemical integrity of DNA precipitated by LaCIs, recovered with

[0148] EDTA (and EGTA) and shown to be a substrate for restriction endonucleases (4A), T4 DNA Ligase

[0149] (4B) and PCR directed amplification (4C and 4D)

[0150] Figure 4A

[0151] Lane Sample

[0152] 1 DNA molecular weight marker

[0153] 10 2 50ng plasmid pUC19 in distilled water

[0154] 3 50ng plasmid pUC19 + Bgll (30mins reaction time)

[0155] 4 50ng plasmid pUC19 recovered from LaCI3precipitation with ImM EDTA

[0156] 5 50ng plasmid pUC19 recovered from LaCI3precipitation with ImM EDTA+ Bgll (30mins reaction time)

[0157] 6 50ng plasmid pUC19 recovered from LaCI3precipitation with ImM EGTA

[0158] 7 50ng plasmid pUC19 recovered from LaCI3precipitation with ImM EGTA+ Bgll (30mins reaction time)

[0159]

[0160] Figure 4B

[0161] Lane Sample

[0162] 1 200ng Bacteriophage Lambda Hind 1 II digest (N3012S)

[0163] DNA digested with 5U Hind 11130 minutes

[0164] 2 200ng Bacteriophage Lambda DNA digested with 5U Hind 111 + T4 DNA Ligase (lOminutes) 3 200ng Bacteriophage Lambda DNA digested with 5U Hind 111 + T4 DNA Ligase (60 minutes) 4 200ng Bacteriophage Lambda DNA digested with 5U Hind 111 + T4 DNA Ligase (120 minutes) 5 200ng Bacteriophage Lambda DNA digested with 5U Hind 111 + T4 DNA Ligase (180 minutes) 6 200ng Bacteriophage Lambda DNA digested with 5U Hindlll, recovered from LaCI3

[0165] precipitation with ImM EDTA + T4 DNA Ligase (10 minutes)

[0166] 7 200ng Bacteriophage Lambda DNA digested with 5U Hindlll, recovered from LaCI3

[0167] precipitation with ImM EDTA + T4 DNA Ligase (60 minutes)

[0168]

[0169] 8 200ng Bacteriophage Lambda DNA digested with 5U Hindlll, recovered from LaCI3precipitation with ImM EDTA + T4 DNA Ligase (120 minutes)

[0170] 9 200ng Bacteriophage Lambda DNA digested with 5U Hindlll, recovered from LaCI3

[0171] precipitation with ImM EDTA + T4 DNA Ligase (180 minutes)

[0172]

[0173] Figure 4C

[0174] Lane Sample

[0175] 1 DNA molecular weight markers

[0176] 2 PCR reaction negative control: no template added

[0177] 3 PCR reaction: pET28a recombinant plasmid positive control template (purified using an NEB Monarch plasmid preparation kit, according to the manufacturer's instructions) and amplified with primers flanking the inserted sequence using NEB's OneTaq master mix, according to the manufacturer's instructions.

[0178] 4 Plasmid recovered from 1ml broth culture of E.coli cells harbouring the recombinant plasmid (Lane 3) used as a template for PCR, as in lane 2. Plasmid was isolated from the cell pellet obtained by centrifugation, resuspended in lOOpI distilled water, followed by cell lysis at 95°C for 5 minutes and recovery of the supernatant material, lpl of supernatant was used as a template

[0179] 5 As in Lane 4, however LaCI3was added to the supernatant to a final concentration of 100pM, the pellet obtained by centrifugation was re-dissolved in 20pl ImMEDTA and lpl used as a template as in lane 2

[0180] 6 As in Lane 4, however LaCI3was added to the supernatant to a final concentration of 200pM, the pellet obtained by centrifugation was re-dissolved in 20pl ImMEDTA and lpl used as a template as in lane 2

[0181] 7 As in Lane 4, however LaCI3was added to the supernatant to a final concentration of 500pM, the pellet obtained by centrifugation was re-dissolved in 20pl ImMEDTA and lpl used as a template as in lane 2

[0182] 8 As in Lane 4, however LaCI3was added to the supernatant to a final concentration of 750pM, the pellet obtained by centrifugation was re-dissolved in 20pl ImMEDTA and lpl used as a template as in lane 2

[0183] 9 As in Lane 4, however LaCI3was added to the supernatant to a final concentration of lOOOpM, the pellet obtained by centrifugation was re-dissolved in 20pl ImMEDTA and lpl used as a template as in lane 2

[0184]

[0185] Figure 4D

[0186] Lane Sample

[0187] 1 DNA molecular weight marker

[0188] 2 PCR primers in 20pl removed from by precipitation with 50pM LaCI3and the supernatant added to a plasmid template and a PCR master mix

[0189]

[0190] PCR primers in 20pl removed from by precipitation with 25pM LaCI3and the supernatant added to a plasmid template and a PCR master mix

[0191] PCR primers in 20pl removed from by precipitation with 5pM LaCI3and the supernatant added to a plasmid template and a PCR master mix

[0192] PCR primers in 20pl removed from by precipitation with lpM LaCI3and the supernatant added to a PCR master mix

[0193] PCR primers in 20pl removed from by precipitation with 50pM LaCI3and the supernatant added to a PCR master mix (no template added)

[0194] PCR primers in 20pl removed from by precipitation with 25pM LaCI3and the supernatant added to a PCR master mix (no template added)

[0195] PCR primers in 20pl removed from by precipitation with 5pM LaCI3and the supernatant added to a PCR master mix (no template added)

[0196] PCR primers in 20pl removed from by precipitation with lpM LaCI3and the supernatant added to a PCR master mix (no template added)

[0197] DNA molecular weight marker

[0198] Control PCR reaction with template, primers and a PCR master mix

[0199]

[0200] Figure 5a

[0201] This figure demonstrates that RNA can be precipitated from cell extracts using LaCl₃

[0202] Lane Sample

[0203] 1 DNA molecular weight marker

[0204] 2 Total RNA isolated following grinding lg dried yeast and resuspending in 1ml 20% (w / v)

[0205] acetonitrile-water. 5pl RNA dissolved in a total volume of distilled water.

[0206] 3 As in lane 2, in the presence of a final concentration of 250pM LaCI3

[0207] 4 As in lane 2, in the presence of a final concentration of 500pM LaCI3

[0208] 5 As in lane 2, in the presence of a final concentration of 1000μM LaCl3

[0209] 6 As in lane 2, in the presence of a final concentration of 1250pM LaCI3

[0210] 7 As in lane 2, in the presence of a final concentration of 1500pM LaCI3

[0211] 8 As in lane 2, in the presence of a final concentration of 2000pM LaCI3

[0212] 9 As in lane 2, in the presence of a final concentration of 2250pM LaCI3

[0213] 10 As in lane 2, in the presence of a final concentration of 2500pM LaCI3

[0214] 11 DNA molecular weight marker

[0215]

[0216] Fig. 5 b shows a schematic diagram of a method of enrichment and depletion of cellular nucleic acids in accordance with the present invention starting with cell extracts.

[0217] Cells 20 are harvested from a culture;

[0218] Step 1 and 2 Cells 20 are lysed (for example by thermolysis, mechanical disruption, chemical or enzymatic disruption) and the insoluble cell debris 22 removed by centrifugation

[0219] Step 3 The supernatant of nucleic acids and soluble cell material is made 0.1-10mM with respect to one of the rare earth salts, typically LaCl₃ and the precipitate forms 25;

[0220] Step 4 Centrifuge to recover nucleic acid (precipitate) 26 and nucleic acid-depleted lysate (supernatant) 28; Step 5 and Step 6 Recover the precipitated nucleic acids 26 and decant the nucleic acid depleted

[0221] lysate 28;

[0222] Step 7 Add chelating agent to resolubilise the nucleic acids;

[0223] Step 8 Nucleic acid is now ready for downstream applications, and

[0224] Step 9 The depleted lysate is now ready for downstream processing.

[0225] Figure 6a

[0226] La3+ions can be immobilised on a strong cation exchanger (SP Sepharose) and used to capture

[0227] DNA from solution, and the DNA subsequently released by the addition of a chelating agent

[0228] (EDTA)

[0229] Lane Sample

[0230] 1 DNA Molecular weight markers

[0231] 2 Supernatant wash 1 (500μL 10 mM Tris-HCl, pH8)

[0232] 3 Elution 1 (50μL 10 mM Tris-HCl, pH8 + 0.1mM EDTA)

[0233] 4 Elution 2 (50μL 10 mM Tris-HCl, pH8 + 0.1mM EDTA)

[0234] 5 Elution 3 (50μL 10 mM Tris-HCl, pH8 + 0.5mM EDTA)

[0235] 6 Elution 4 (50μL 10 mM Tris-HCl, pH8 + 0.5mM EDTA)

[0236]

[0237] 10μl (100ng) DNA markers (NEB) mixed with 50μl SP Sepharose XL (Cytiva: strong cation

[0238] exchanger) loaded with LaCl₃ to generate an immobilised La3+solid phase (according to the

[0239] manufacturer's instructions). The suspension was gently agitated at room temperature for

[0240] 30minutes, after which it was centrifuged at 10 000rpm and 20μl of the supernatant loaded onto a

[0241] 1% agarose gel. The beads were then successively mixed with increasing concentrations of EDTA in Tris Buffer (as indicated), the supernatants recovered and analysed by agarose gel electrophoresis. Recovered nucleic acids are identified in lane 5 using bracket (a).

[0242] Fig. 6b shows a schematic workflow illustrating the steps in the enrichment and depletion of cell-free nucleic acids using La3+ions immobilised on NTA-derivatised agarose ferromagnetic beads. Step 1 Cells in liquid medium 30 are pelleted from a given medium (cultured, tissue-fluid-derived or preserved cells, for example)

[0243] Step 2 The cell-free supernatant containing cell-free nucleic acid 32 is transferred to a clean tube. (Pelleted cells 34 may be used for cell lysis and subsequent nucleic acid enrichment and / or depletion);

[0244] Step 3 Typically La3+beads 36 (magnetic beads charged with rare earth ions) are added as 10% supernatant volume, and mixed gently for 10-20 minutes at room temperature;

[0245] Step 4 The beads 36 are captured by means of a magnet 38 or magnetic sample rack;

[0246] Step 5 The supernatants 40 is removed and the beads 36 are washed with a non-phosphate buffer, eg 10mM Tris at physiological pH;

[0247] Step 6 The captured nucleic acid is released by the addition of a chelating agent and is now available for downstream processing, and

[0248] Step 7 For depletion, the supernatant 40 is now available for downstream processing. Figure 7

[0249] Demonstration of the biochemical activity / integrity of RNA isolated by precipitation with LaCl₃

[0250] followed by recovery with 1mM EDTA. The RNA was used as a substrate for a quantitative RTPCR

[0251] experiment and the product yield and amplification profiles were shown to be dependent on the

[0252] presence and relative concentration of the RNA template.

[0253] RTQPCR sample Description

[0254] 1 Total RNA

[0255] 2 Total RNA (10 x dilution)

[0256] 3 Total RNA (100 x dilution)

[0257] 4 RNA precipitated with 50μM LaCl3, recovered with 1mM EDTA

[0258] 5 RNA remaining in the supernatant after 50μM LaCl3precipitation

[0259] 6 RNA precipitated with 250μM LaCl3, recovered with 1mM EDTA

[0260] 7 RNA remaining in the supernatant after 250μM LaCl3precipitation

[0261] 8 RNA precipitated with 500μM LaCl3, recovered with 1mM EDTA

[0262] 9 RNA remaining in the supernatant after 500μM LaCl3precipitation

[0263] 10 Control: No RNA added

[0264] 11 Control: No Reverse Transcriptase added

[0265]

[0266] Total RNA (100μl) was isolated from 0.5ml of a mid-log phase culture of Saccharomyces cerevisiae

[0267] using a Monarch Total RNA purification kit (NEB), following the manufacturer's instructions. 5pl of

[0268] isolated RNA was precipitated with LaCl₃ at concentrations of 50μM, 250μM or 500μM, followed

[0269] by recovery with 1mM EDTA pH 8. Each RNA sample was then used to feed an RTQPCR reaction

[0270] using primers specific to the sequence encoding the ACT1 mRNA, which flank the intron-encoding sequence present in the genomic sequence (thereby ensuring amplification is templated by mRNA

[0271] and not genomic DNA). The reaction was initiated using a Luna probe master mix and enzymes

[0272] (NEB) according to the manufacturer's instructions. The reactions were all performed using an

[0273] AriaMx Real-time PCR System (Agilent), in triplicate. The Cq values were determined using the on-

[0274] board software and results are presented in the form of a box plot.

[0275] Table 3

[0276] Salt Concentration (pM) required to precipitate lOOng DNA

[0277] ScCI350

[0278] YCI350

[0279] LaCI350

[0280] La(NO3)350

[0281] Nd(NO3)350

[0282] Sm(NO3)350

[0283] HoCl350

[0284] ErCI350

[0285] YbCI350

[0286] TbCI350

[0287] CeCI3250

[0288] PrCI3250

[0289] Dy(NO3)3250

[0290] TmCI3250

[0291] EuCl3250

[0292] LuCl3250

[0293]

[0294] GdCl350

[0295] Agarose gel electrophoresis was used to determine the minimum concentration required to

[0296] precipitate 100ng of bacteriophage lambda DNA (as shown in Figure 1). Results obtained with

[0297] GdCIswere irreproducible and may relate to an unknown reaction of this salt with DNA. Table 3

[0298] indicates the capacity of soluble lanthanoid salts to precipitate DNA at concentrations <lmM. Table 4

[0299] Chelator Minimum concentration (pM) required to recover lOOng pUC19 plasmid DNA

[0300] following precipitation with 100 μM LaCl3

[0301] EDTA 500

[0302] EGTA 1

[0303] Citrate 500

[0304] NTA 10

[0305]

[0306] In each experiment, 100ng of plasmid pUC19 added to a final concentration of 1ng / μl in a solution of 100μM LaCl₃. After 5 minutes, each suspension was vortexed thoroughly, the contents centrifuged at 15,000rpm for 2 minutes, in a bench-top centrifuge, and the supernatants discarded. To each pellet was then added 20pl of a solution of the sodium salt of the above chelators, at concentrations ranging from 1-1000μM. Each sample was then applied to an agarose gel containing SYBR green DNA detection dye, electrophoresed for 40 minutes (as in Fig. 1) and the minimum concentration required to recover >90% of the nucleic acid was determined. Table 4 shows the concentrations of a range of metal ion chelators required to solubilise a fixed amount of DNA precipitated by the addition of 50μM LaCl₃ Table 5

[0307] LaCI3concentration (|1M) Transformation efficiency (cfu / pg Monarch purified, plasmid DNA,

[0308] with NEB BL21(DE3) competent cells)

[0309] 0 1 x 107

[0310] 50* 5 x 106

[0311] 100 5 x 106

[0312] 1000 0

[0313]

[0314] *The nucleotide sequences of triplicate samples of plasmids recovered from this transformation reaction were obtained independently, via the Plasmidsaurus service: there were no variations in sequence compared with plasmids obtained from the LaCl₃ free transformants. Table 5 demonstrates the biocompatibility of plasmid DNA purified using LaCl₃, as illustrated by the negligible impact of low concentrations of LaCl₃ on bacterial transformation efficiency and the integrity of the recovered plasmids, as determined by nucleotide sequencing.

[0315] Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the description and dependent claims. It should be understood, however, that the description, dependent claims, and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the scope of the disclosed invention will become readily apparent to those skilled in the art from reading the specification.

Claims

Claims1. A method of depleting and / or enriching nucleic acid from a sample comprising the steps of:(a) contacting a sample containing nucleic acid with a rare earth metal ion having a metal phosphate Ksp of less than 10-24to form a metal nucleic acid precipitate and a supernatant;(b) separating the precipitate and the supernatant; and(c) collecting the metal nucleic acid precipitate and / or supernatant.

2. The method of claim 1 wherein the depleted and / or enriched nucleic acid at least substantially retains its function.

3. The method as claimed in claim 1 or 2 wherein the rare earth metal ion is present in step a) at a concentration of about lOmM or less.

4. The method of claim 3 wherein the rare earth metal ion is present in step a) at a concentration of about ImM or less.

5. The method of claim 4 wherein the rare earth metal ion is present in step a) at a concentration of about 25 pm to about ImM.

6. The method of claim 4 wherein the rare earth metal ion is present in step a) at a concentration of about 25 pm to about 100 pm.

7. The method of claim 6 wherein the rare earth metal ion is present in step a) at a concentration of about 40 pm to about ImM.

8. The method of claim 7 wherein the rare earth metal ion is present in step a) at a concentration of about 40 pm to about 250 pm.

9. The method of claim 8 wherein the rare earth metal is present in step a) at a concentration of about 50 pm.

10. The method as claimed in any one of the previous claims wherein the rare earth metal ion is an ion derived from a lanthanoid.

11. The method as claimed in any one of the previous claims wherein the rare earth metal is an ion derived from any one or more selected from the group comprising: Scandium, (Sc), Yttrium (Y), Lanthanum (La), Cerium (Ce), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), and Lutetium (Lu).

12. The method as claimed in any one of the previous claims wherein the rare earth metal ion is derived from lanthanum chloride.

13. The method as claimed in any one of the previous claims further comprising the subsequent step of eluting the nucleic acid from the precipitate by lowering the pH and / or the addition of a chelating agent.

14. The method of claim 13 wherein the chelating agent is selected from the group comprising: EDTA, EGTA, NTA, citrate.

15. The method of any one of the previous claims wherein the eluted nucleic acid retains its function / bioactivity.

16. The method of any one of the previous claims wherein the recovered nucleic acid is used in a subsequent biological process.

17. The method of any one of the previous claims wherein metal nucleic acid precipitate is collected by means of a magnet.

18. The method of any one of the previous claims wherein an acid is used for lowering the pH to release the nucleic acid from the precipitate.

19. The method of any one of the previous claims wherein the rare earth ion is immobilized on a solid substrate.

20. The method of any one of the previous claims wherein the chelating agent is immobilized on a solid substrate.

21. The method of any one or more of the previous claims wherein one or more coprecipitation agents is used in step a).

22. The method of any one of the previous claims wherein the nucleic acid is ribonucleic acid (RNA), deoxyribonucleic acid (DNA) or mixtures thereof.

23. The method of claim any one of the previous claims wherein prior to or concomitantly with step (a), said sample is contacted with a lytic agent.

24. The method of claim 23 wherein said lytic agent comprises one or more of ionic and nonionic detergents, hypotonic solutions of salts, protease, chaotropic agents or solvents, grinding tissue or adding other lytic enzymes.

25. The method of any one of the previous claims wherein the method is performed at room temperature and pressure.

26. The method of any one or more of the previous claims wherein one or more of the method steps is automated.

27. A kit for depleting and / or enriching nucleic acid according to the method of any one or more of the previous claims, which comprisesi) one or more rare earth metal ions; andii) optionally one or more wash solutions andiii) one or more elution solutions comprising an acid and / or chelating agent.

28. The kit of claim 27 wherein said rare earth metal ion is immobilised on a solid phase or said chelating agent is immobilised on a solid phase.

29. The kit as claimed in claim 27 or 28 wherein the rare earth metal ion is derived from lanthanum chloride.

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