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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

• the present invention refers to a method for isolating nucleic acids from a nucleic acid-containing sample. More specifically, the present invention relates to a method for sequentially isolating DNA and RNA from the same nucleic acid-containing sample. Furthermore, the present invention refers to kit for sequentially isolating DNA and RNA from the same nucleic acid-containing sample.
• nucleic acids such as DNA and RNA
• Procedures involving the Isolation and/or concentration of nucleic acids continue to play a crucial roie in biotechnology.
• Early methods of isolating nucleic acids involve a series of extractions using organic solvents, followed by ethanol precipitation and dialysis of the nucleic acids. These methods are relatively laborious and often result in a low nucleic acid yield.
• RNA isolation procedures show some degree of selectivity between DNA and RNA.
• the selective isolation of RNA is performed either under acidic conditions on a silica solid phase (US 5,990,302) or under chaotropic conditions in the presence of an alcohol on a silica solid phase (WO 95/01359). In the latter case, normally a low selectivity for RNA over DNA is observed, and a DNA digest step is thus included to obtain pure RNA.
• some methods for the selective isolation of DNA are known from the art.
• RNA and DNA are isolated simultaneously from one nucleic acid-containing sample.
• These methods comprise isolation of the total nucleic acids on a silica solid phase under chaotropic conditions and excluding the DNA digest step as described above.
• the problem underlying the present invention is to overcome the disadvantageous arising from the methods known in the art and to provide a method which allows for the sequential isolation of DNA and RNA from the same sample, i.e. the sequential isolation of DNA and RNA in different eluates from the same nucleic acid-containing sample.
• the problem is solved by the method according to the present invention which allows for sequentially isolating DNA and RNA from the same nucleic acid-containing sample, comprising the steps of:
• step (a) adding a chaotropic salt to a final concentration of from 1 to 4.5 M to a nucleic acid-containing sample, (b) following step (a), adding an alcohol to a final concentration of from 13 to 70 %(v/v) to the solution of (a),
• step (c) bringing the solution of step (b) into contact with a functional surface, maintaining this contact for a particular time period, breaking the contact between the solution of step (b) and the functional surface,
• step (d) bringing the DNA-depleted solution of step (c) into contact with a functional surface, maintaining this contact for a particular time period, breaking the contact between the solution of step (c) and the functional surface,
• step (c) is less than half of the time period of step (d).
• DNA and RNA both are able to bind to a functional surface, e.g. a silica surface, under suitable conditions.
• DNA and RNA bind with different kinetics to a functional surface, e.g. a silica surface, in the presence of both a particular concentration of a chaotropic salt and a particular concentration of an alcohol.
• the functional surface utilized in step (d) is of the same kind as the functional surface utilized in step (c).
• the method according to the invention is, thereby, less laborious and easier to handle.
• the binding conditions for DNA and RNA do not have to be changed between step (c) and step (d) due to the same kind of solid support utilized in step (c) and step (d).
• the present invention provides a method for sequentially isolating DNA and RNA from the same nucleic acid-containing sample, comprising the steps of:
• step (b) following step (a), adding an alcohol to a final concentration of from 13 to 70 %(v/v) to the solution of (a),
• step (c) bringing the solution of step (b) into contact with a functional surface, maintaining this contact for a particular time period, breaking the contact between the solution of step (b) and the functional surface,
• step (d) bringing the DNA-depleted solution of step (c) into contact with a functional surface, maintaining this contact for a particular time period, breaking the contact between the solution of step (c) and the functional surface,
• step (c) is less than half of the time period of step (d).
• the functional surface utilized in step (d) is of the same kind as the functional surface of step (c).
• the method according to the invention is, thereby, less laborious and easier to handle.
• the binding conditions for DNA and RNA do not have to be changed between step (c) and step (d) due to the same kind of solid support utilized in step (c) and step (d).
• a functional surface useful in the present invention is either a surface comprising carboxylic acid groups or, preferably, the functional surface is a silica surface.
• the functional surface is provided in bead form. If the functional surface is provided in bead form, preferably the functional surface comprises a plurality of beads.
• these beads are magnetic beads, i.e. the beads are magnetically attractable.
• all sorts of magnetically attractable beads are useful in the present invention, e.g. paramagnetic beads, superparamagnetic beads, ferrimagnetic beads and/or ferromagnetic beads.
• the number of beads utilized in step (c) is less than half of the number of beads utilized in step (d), thereby advantageously reducing the material consumption and, accordingly, the material costs. Therefore, the method of the invention is particularly suitable in high throughput screening procedures by reducing, e.g., the necessary sample amount/volume, time and costs.
• the number of beads utilized in step (c) is less than 1/5 of the number of beads utilized in step (d) and in an even more preferred embodiment the number of beads utilized in step (c) is less than 1/10 of the number of beads utilized in step (d).
• the term 'DNA' comprises all imaginable types of DNA of any length, e.g. genomic DNA, plastidial DNA, plasmids, cosmids, phasmids, reverse transcription products, PCR products, oligonucleotides or the like.
• the term 'DNA' may also comprise a mixture of different DNA types, e.g. total DNA from a natural source, e.g. cells.
• 'RNA' comprises all imaginable types of RNA of any length, e.g. mRNA, tRNA, rRNA, small nuclear RNA, ribozymes or the like.
• the term 'RNA' may also comprise a mixture of different RNA types, e.g. total RNA from a natural source, e.g. cells.
• the term 'sequential isolation stands for the separated isolation of at first DNA followed by RNA from the same nucleic acid-containing sample. Therefore, DNA and RNA are isolated separately but in a continuous process and from the same nucleic acid-containing sample. Thus, the present invention allows for a comparatively low sample amount/volume for the isolation of DNA and RNA in separated fractions from one nucleic acid-containing sample.
• the source of the nucleic acids contained in the nucleic acid-containing sample may be any imaginable source. It may either be a natural source, e.g. from cells or tissue or the like, or an artificial source, e.g. a PCR product or the like. If the source is a natural source, it is regardless of which kind the natural source is, i.e. the source may be procaryotic or eucaryotic, the source may be single cells or tissue or even subcellular fractions.
• the nucleic acid-containing sample has to be an aqueous solution, e.g. a cell lysate, or has to be brought into an aqueous solution by addition of a chaotropic salt solution according to step (a) or any suitable solvent. Solvents suitable to bring nucleic acids into solution are well known to those skilled in the art.
• the chaotropic salt is added in step (a) in form of a solution and can advantageously be used to lyse the source of the nucleic acids, e.g. cells or tissue. In this case an additional sufficient incubation time is needed to allow the cells to lyse.
• the required conditions to lyse the source of the nucleic acids i.e. incubation time, temperature etc., are well known to a person skilled in the art and can easily be adapted to the method according to the invention.
• the chaotropic salt added to the nucleic acid-containing sample in step (a) is selected from urea, sodium iodide, potassium iodide, sodium permanganate, potassium permanganate, sodium perchlorate, potassium perchlorate, sodium chlorate, potassium chlorate, guanidinium hydrochloride, guanidinium isothiocyanate, guanidinium thiocyanate, hexamine cobalt chloride, tetramethyl ammonium chloride, alkyltrimethyl ammonium chloride, tetraethyl ammonium chloride, tetramethyl ammonium iodide, alkyltrimethyl ammonium iodide, tetraethyl ammonium iodide or is a mixture thereof.
• alkyl represents a branched or unbranched hydrocarbon radical having 1 to 20 carbon atoms.
• the chaotropic salt is added to the nucleic acid-containing sample as a solution of suitable concentration.
• suitable solvent e.g. water or a buffer system
• Suitable solvents or buffer systems according to the present invention are obvious to a skilled person.
• the chaotropic salt can be added as a solid. In the latter case, it is required that the nucleic acid-containing sample is available as a solution.
• the chaotropic salt is added in step (a) to a final concentration in a range of from 1 to 4.5 M.
• the chaotropic salt is added in step (a) to a final concentration in a range of from 1.2 to 3.5 M and more preferably in a range of from 1.5 to 3 M.
• the term 'final concentration' for the purpose of the present invention stands for the concentration of a substance, i.e. the chaotropic salt added in step (a) and the alcohol added in step (b), after adding the chaotropic salt in step (a) and after adding the alcohol in step (b) but before step (c) of the present invention.
• the alcohol added in step (b) is selected from methanol, ethanol, n-propanol, iso- propanol or is a mixture thereof. Thereby, the alcohol is added pure or diluted in a suitable solvent, e.g. water, to a suitable concentration.
• a suitable solvent e.g. water
• the alcohol is added in step (b) to a final concentration in a range of from 13 to 70 %(v/v). In a preferred embodiment, the alcohol is added in step (b) to a final concentration in a range of from 25 to 60 %(v/v) and more preferably in a range of from 30 to 50 %(v/v).
• An essential feature of the present invention is that the different binding kinetics of RNA and DNA in the presence of a chaotropic salt and an alcohol to a functional surface are utilized. Therefore, the time period of step (c) is less than half of the time period of step (d), i.e. the kinetics for the DNA binding is much faster than the kinetics for the RNA binding under suitable conditions according to the invention. Therefore, for the binding of DNA to the functional surface a significant shorter incubation time is needed as compared to the incubation time needed for the binding of RNA to the functional surface.
• the time period of step (c) is less than 1/10 of the time period of step (d) and more preferably the time period of step (c) is less than 1/20 of the time period of step (d).
• the time period in step (c) is in a range of from 5 seconds to 60 seconds and the time period in step (d) is 30 seconds or more, depending on the time period in step (c).
• the time period in step (d) has no strict upward boundaries, but is limited upwards to an incubation time which appears suitable to a person skilled in the art, i.e. either a degradation of the RNA or an unhelpful prolongation of the method according to the invention should be avoided.
• the method of the present invention can be performed at any suitable temperature. A suitable temperature for such a method is obvious to a person skilled in the art.
• the preferred temperature range for the present invention is room temperature (18°C to 25°C).
• the method is performed in an automated process. Due to the fact that similar methods utilizing, e.g., magnetic beads are well known from the art as automated processes, the method of the invention can easily be adapted to an automated process.
• RNA bound to the functional surface in step (c) is a DNA-rich fraction.
• the DNA has to be further purified.
• the RNA isolation step (d) The solution obtained by step (c) after breaking the contact between the solution and the functional surface is DNA- depleted but is not free of DNA. Therefore, during the prolonged incubation time in step (d) a small amount of DNA will bind the functional surface.
• the RNA bound to the functional surface in step (d) is a RNA-rich fraction.
• the RNA has to be further purified.
• the further purification steps as mentioned above may be any purification procedures known from the art and suitable for a purification of nucleic acids bound to a functional surface according to the present invention.
• the further purification normally comprises at least one washing step, an optional nuclease treatment and an elution step.
• step (c) may comprise at least the steps of:
• step (c) washing the DNA bound to the functional surface of step (c) after breaking the contact between the solution of step (b) and the functional surface with a suitable solution, (2) eluting the DNA from the functional surface using a suitable solution,
• step (3) prior to, simultaneously with or following step (1) or step (2), performing a RNase treatment under suitable conditions.
• step (d) may in general comprise at least the steps of:
• step (d) washing the RNA bound to the functional surface of step (d) after breaking the contact between the DNA-depleted solution of step (c) and the functional surface with a suitable solution
• step (3) prior to, simultaneously with or following step (1) or step (2), performing a
• a suitable solution comprising either a high concentration of chaotropic salt, e.g. 7 M guanidinium hydrochloride, or a high concentration of a suitable alcohol, e.g. ethanol at 65 to 80 % (v/v), or a suitable organic solvent, the nucleic acids being insoluble in this organic solvent.
• a suitable solution compositions and performance of such washing steps are known from the state of the art. At least one washing step may be performed or the washing steps may be performed in a number seeming suitable to a person skilled in the art.
• An exemplary and non-limiting procedure is to perform one or two washing steps with solution comprising a high concentration of chaotropic salt, e.g. 5.4 M guanidinium thiocyanate, to remove biological contaminants followed by at least two washing steps with a solution comprising a high concentration of an alcohol, e.g. 80% (v/v) ethanol, to remove the chaotropic salt.
• a nuclease treatment may be performed.
• the nucleic acids are normally eluted from the functional surface to perform a nuclease treatment.
• the elution from the functional surface is achieved by, e.g., resuspending the functional surface in a low salt solution or water.
• Any suitable enzyme and any suitable solution for the nuclease treatment may be utilized. Such enzymes and solutions are well known to those skilled in the art.
• the nuclease treatment can be performed at any stage of the further purification as mentioned above.
• the nucleic acids may be rebound to the functional surface from which they were eluted after the nuclease treatment by, e.g., changing the concentration(s) of the substance(s) of content.
• the functional surface for binding the nucleic acids is steps (c) and/or (d) has not inevitably to be of the same kind as the functional surface for rebinding the nucleic acid.
• the nuclease may be inactivated, e.g. by heat or any other suitable measure, and the nucleic acids may be used for other purposes without being rebound to the functional surface, e.g. precipitating the nucleic acids by addition of an alcohol followed by collecting the nucleic acids by, e.g., centrifugation. Suitable methods are well known to those skilled in the art.
• the present invention provides a kit for sequentially isolating DNA and RNA from the same nucleic acid-containing sample according to the present invention.
• the kit comprises at least a functional surface according to the invention and/or a chaotropic salt and/or an alcohol.
• the chaotropic salt may be part of the kit as, e.g., a solid or as a stock solution or as a ready-to-use solution.
• the alcohol may be part of the kit as, e.g., a stock solution or as a ready-to- use solution.
• the kit furthermore comprises substances and/or devices allowing for a further purification of the isolated DNA and RNA according to one of the several different methods known in the art. Examples
• Crossing point (Ct) values from real-time PCR were used as a measure for the concentration of DNA and RNA in the eluted fractions of nucleic acids.
• Ct Crossing point
• GtB-CtA concentration B] E " .
• a frozen cell pellet of 1 x 10 6 HL60 cultured cells was lysed in 100 ⁇ Solution A.
• the lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 300 ⁇ Solution A were added and gently mixed. To this solution, 300 ⁇ 96 %(v/v) ethanol were added. The final concentrations were:
• RNA isolation comprises the steps of:
• step (b) 60 ⁇ of a magnetic particle suspension being of the same kind as the magnetic particle suspension utilized in step (a) were added to the partially DNA-depleted lysate from step (a) and incubated for 3 minutes. Subsequently, the ferrimagnetic particles were removed from the lysate by a magnet (fraction 2) and the remaining solution was discarded.
• a frozen cell pellet of 1 x 10 6 HL60 cultured cells was lysed in 100 I Solution A.
• the lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 500 ⁇ Solution A were added and gently mixed. To this solution, 100 ⁇ 96 %(v/v) ethanol were added. The final concentrations were:
• step (a) 6 mg of ferrimagnetic particles were suspended in 20 ⁇ of an aqueous solution comprising a composition according to the final composition of the lysate as displayed above (13.7 %(v/v) ethanol, 3.0 M guanidinium thiocyanate, 21.5 mM trisodium citrate).
• a frozen cell pellet of 1 x 10 6 HL60 cultured cells was lysed in 100 ⁇ Solution A.
• the lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 100 ⁇ Solution A were added and gently mixed. To this solution, 500 ⁇ 96 %(v/v) ethanol were added. The final concentrations were:
• step (a) 6 mg of ferrimagnetic particles were suspended in 20 ⁇ of an aqueous solution comprising a composition according to the final composition of the lysate as displayed above (68.6 %(v/v) ethanol, 1.0 M guanidinium thiocyanate, 7.2 mM trisodium citrate).
• a frozen cell pellet of 1 x 10 6 HL60 cultured cells was lysed in 100 /I Solution A.
• the lysate was further homogenized by 5 times passing a 23G syringe.
• 100 /I Solution A were added and gently mixed.
• 500 ⁇ 100 %(v/v) iso-propanol were added. The final concentrations were:
• step (a) 6 mg of ferrimagnetic particles were suspended in 20 ⁇ of an aqueous solution comprising a composition according to the final composition of the lysate as displayed above (71.5 %(v/v) iso-propanol, 1.0 M guanidinium thiocyanate, 7.2 mM trisodium citrate).
• a frozen cell pellet of 1 x 10 6 HL60 cultured cells was lysed in 100 /I Solution B.
• the lysate was further homogenized by 5 times passing a 23G syringe.
• 300 ⁇ Solution B were added and gently mixed.
• 300 ⁇ 96 %(v/v) ethanol were added. The final concentrations were:
• step (b) 60 ⁇ of a magnetic particle suspension being of the same kind as the magnetic particle suspension utilized in step (a) were added to the partially DNA-depleted lysate from step (a) and incubated for 3 minutes. Subsequently, the ferrimagnetic particles were removed from the lysate by a magnet (fraction 2) and the remaining solution was discarded.
• Crossing point (Ct) values from real-time PCR were measured both with and without a preceding reverse transcription. Without reverse transcription the Ct value is a measure of the DNA concentration. Including a preceding reverse transcription the Ct value is a measure of the total amount of RNA and DNA. The difference between the Ct value with and without preceding reverse transcription is usually referred to as ⁇ Ct. ⁇ Ct is commonly used as a measure for RNA fraction of total nucleic acid. Larger ⁇ Ct values identify higher RNA fractions of total nucleic acids. Results
• a frozen cell pellet of 1 x 10 6 HL60 cultured cells was lysed in 100 ⁇ Solution A.
• the lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 300 ⁇ Solution A were added and gently mixed. To this solution, 300 ⁇ 100 %(v/v) methanol were added. The final concentrations were:
• step (a) 6 mg of ferrimagnetic particles were suspended in 20 ⁇ of an aqueous solution comprising a composition according to the final composition of the lysate as displayed above (42.8 %(v/v) methanol, 2.0 M guanidinium thiocyanate, 14.4 mM trisodium citrate).
• a frozen cell pellet of 1 x 10 6 HL60 cultured cells was lysed in 100 ⁇ Solution A. Subsequently, 100 ⁇ Solution A were added and gently mixed. To this solution, 150 ⁇ 96 %(v/v) ethanol were added. The final concentrations were:
• the subsequent isolation of nucleic acids comprises the steps of:
• step (b) 80 ⁇ of a magnetic particle suspension being of the same kind as the magnetic particle suspension utilized in step (a) were added to the partially DNA-depleted lysate from step (a) and incubated for 2 minutes.
• ferrimagnetic particles were removed from the lysate by a magnet (fraction 2) and the remaining solution was discarded.

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