EP1633869A1 - Method for sequentially isolating dna and rna from the same nucleic acid-containing sample - Google Patents

Method for sequentially isolating dna and rna from the same nucleic acid-containing sample

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Publication number
EP1633869A1
EP1633869A1 EP04739565A EP04739565A EP1633869A1 EP 1633869 A1 EP1633869 A1 EP 1633869A1 EP 04739565 A EP04739565 A EP 04739565A EP 04739565 A EP04739565 A EP 04739565A EP 1633869 A1 EP1633869 A1 EP 1633869A1
Authority
EP
European Patent Office
Prior art keywords
functional surface
dna
solution
rna
time period
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP04739565A
Other languages
German (de)
French (fr)
Inventor
Evy H. Reitan
Arne Deggerdal
Vidar Skagestad
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qiagen GmbH
Qiagen AS
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Qiagen GmbH
Qiagen AS
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Filing date
Publication date
Application filed by Qiagen GmbH, Qiagen AS filed Critical Qiagen GmbH
Publication of EP1633869A1 publication Critical patent/EP1633869A1/en
Ceased legal-status Critical Current

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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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Abstract

The present invention refers to a method for isolating nucleic acids from a nucleic acid-containing sample. More specifically, it 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.

Description

METHOD FOR SEQUENTIALLY ISOLATING DNA AND RNA FROM THE SAME
NUCLEIC ACID-CONTAINING SAMPLE
BACKGROUND OF THE INVENTION
Field of the Invention 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.
Description of the Related Art
Procedures involving the Isolation and/or concentration of nucleic acids such as DNA and RNA 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.
Later methods have taken advantage of the fact that nucleic acids are bound to silica surfaces in the presence of a chaotrope. This was first described for diatomeous earth and for silicon dioxide particles (US 5,234,809). More recently, the use of magnetically attractable particles with silica surfaces have been described (US 6,027,945; US 6,582,922; WO 04/003231), simplifying the handling of the nucleic acid isolation systems.
Some methods are known from the art, in which the silica surface/chaotropic salt- based nucleic acid 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. Likewise, some methods for the selective isolation of DNA are known from the art. Additionally, some methods are described in the state of the art which allow for the isolation of total nucleic acids, which means that both, 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.
Presently, no methods are known from the art which allow for the sequential isolation of nucleic acids from the same sample, i.e. the sequential isolation of DNA and RNA in different eluates from the same nucleic acid-containing sample. However, the current methods known from the art leave the option to perform at first a DNA isolation step and then in a separated step a RNA isolation step with an additional amount of the nucleic acid-containing sample, or vice versa. There are several disadvantages of such an approach, e.g. it is time consuming, immoderate laborious and a high sample volume/amount is required. Especially when only low amounts of nucleic acid-containing sample are available, e.g. human tissue or the like, new technologies to circumvent these limitations are imperatively needed.
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.
BRIEF SUMMARY OF THE INVENTION
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:
(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),
(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,
(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,
wherein the time period of step (c) is less than half of the time period of step (d).
As known from the art, DNA and RNA both are able to bind to a functional surface, e.g. a silica surface, under suitable conditions. Surprisingly, it has been found that 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. These different properties allow for the sequential isolation of DNA followed by the isolation of RNA from the same nucleic acid- containing sample, i.e. DNA and RNA are isolated in two different steps in two different eluates in a continuous process from the same nucleic acid-containing sample. Advantageously, the present invention provides a relatively fast method, which is easy to handle, and wherein a comparatively low sample volume/amount is required for the isolation of DNA and separated from that the isolation of RNA.
In a preferred embodiment of the invention, the functional surface utilized in step (d) is of the same kind as the functional surface utilized in step (c). Advantageously, 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). DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for sequentially isolating DNA and RNA from the same nucleic acid-containing sample, comprising the steps of:
(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),
(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,
(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,
wherein the time period of step (c) is less than half of the time period of step (d).
In a preferred embodiment of the invention, the functional surface utilized in step (d) is of the same kind as the functional surface of step (c). Advantageously, 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. In a preferred embodiment of the present invention, 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. In a more preferred embodiment, these beads are magnetic beads, i.e. the beads are magnetically attractable. Thereby, all sorts of magnetically attractable beads are useful in the present invention, e.g. paramagnetic beads, superparamagnetic beads, ferrimagnetic beads and/or ferromagnetic beads. In a preferred embodiment of the invention, 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. In a more preferred embodiment, 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).
In the present invention 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.
In the present invention the term '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. According to the invention, 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.
For some embodiments, it is essential to lyse the source of nucleic acids, e.g. cells, tissue or the like, to obtain a nucleic acid-containing sample which is suitable for use in the present invention. In one embodiment of the invention, 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. In the present invention, alkyl represents a branched or unbranched hydrocarbon radical having 1 to 20 carbon atoms.
Preferably, the chaotropic salt is added to the nucleic acid-containing sample as a solution of suitable concentration. Every suitable solvent, e.g. water or a buffer system, can be applied to solubilize the chaotropic salt. Suitable solvents or buffer systems according to the present invention are obvious to a skilled person. Alternatively, 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. In a preferred embodiment, 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.
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. In a preferred embodiment, 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). Contemporaneously to the above mentioned, 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).
In another embodiment of the present invention, 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.
The much faster kinetics of DNA binding implies that RNA will also bind to the functional surface in step (c), even if only small amounts of RNA will bind to the functional surface. Therefore, the DNA bound to the functional surface in step (c) is a DNA-rich fraction. To obtain RNA-free DNA, the DNA has to be further purified. Vice versa, the same applies for 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. Thus, the RNA bound to the functional surface in step (d) is a RNA-rich fraction. To obtain DNA-free RNA, 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.
In general, the further purification of the DNA bound to the functional surface during step (c) may comprise at least the steps of:
(1) 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,
(3) prior to, simultaneously with or following step (1) or step (2), performing a RNase treatment under suitable conditions.
Accordingly, the further purification of the RNA bound to the functional surface during step (d) may in general comprise at least the steps of:
(1) 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,
(2) eluting the RNA from the functional surface using a suitable solution,
(3) prior to, simultaneously with or following step (1) or step (2), performing a
DNase treatment under suitable conditions.
Several methods are known from the art to further purify the nucleic acids obtained by the present invention. Some non-limiting examples for purification procedures are given below.
During the washing procedure as mentioned, in step (1), the nucleic acids normally stay bound to the functional surface. Therefore, a suitable solution is needed 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. 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. To make sure that the DNA isolated by the method according to the invention is free of RNA and, vice versa, that the RNA isolated by the method according to the invention is free of DNA, a nuclease treatment may be performed. As no nuclease is able to work in the solutions normally used to keep the nucleic acids bound to the functional surface during the washing procedure, 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. Thereby, 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. Similarly, 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.
In another aspect, 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. For instance, 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. For instance, the alcohol may be part of the kit as, e.g., a stock solution or as a ready-to- use solution. In another embodiment, 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
The following non-limiting examples are provided for the purpose of illustration. The following solutions are utilized in the examples:
Solution A (100 g): 37.6 g guanidinium thiocyanate
0.7 g trisodium citrate 61.7 ml water
Solution B (100 g): 58.3 g guanidinium hydrochloride
41.7 ml water
Example 1
Estimation of concentration ratios based on real-time PCR
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. To measure values for RNA, a DNase treatment step was included in the protocol and reverse transcription was performed prior to real-time PCR. To measure values for DNA, both the DNase treatment and the reverse transcription were omitted. The concentration
-Ct at the outset of PCR is proportional to E , wherein E is the amplification efficiency.
Hence, if two reactions A and B have the same E, then [start concentration A]/[start
GtB-CtA concentration B] = E " . The amplicon used (here: β-actin) was shown to have an efficiency of 1.9 under the chosen experimental conditions. Therefore, [DNA concentration fraction 1/ DNA concentration fraction 2] = 1.9 lon ' and [RNA concentration fraction 2/ RNA concentration fraction 1] = 1.9 ^ rac lon r rac ι , wherein the data from runs without DNase treatment and reverse transcription were used to calculate the first fraction as a measure of enrichment of DNA in fraction 1 and the data from runs including DNase treatment and reverse transcription were used to calculate the second fraction as a measure of enrichment of RNA in fraction 2.
Experimental setup for the RNA assay
A frozen cell pellet of 1 x 106 HL60 cultured cells was lysed in 100 μ\ Solution A. The lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 300 μ\ Solution A were added and gently mixed. To this solution, 300 μ\ 96 %(v/v) ethanol were added. The final concentrations were:
41.1 %(v/v) ethanol
2.0 M guanidinium thiocyanate
14.4 mM trisodium citrate
The subsequent isolation of RNA comprises the steps of:
(a) 6 mg of ferrimagnetic particles according to WO 04/003231 were suspended in 20 μ\ of an aqueous solution comprising a composition according to the final composition of the lysate as displayed above (41.1 %(v/v) ethanol, 2.0 M guanidinium thiocyanate, 14.4 mM trisodium citrate). This suspension was added to the lysate, incubated for 10 seconds and the ferrimagnetic particles were removed from the lysate by a magnet (fraction 1).
(b) 60 μ\ 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.
(c) The fraction 1 and fraction 2 ferrimagnetic particles were given parallel identical treatments using the QIAGEN MagAttract RNA kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The optional DNase treatment was performed with both fractions of ferrimagnetic beads.
Experimental setup for the DNA assay
Experiments were performed as described above except for the optional DNase treatment included in the protocol of the QIAGEN MagAttract RNA kit (see step (c)) which was not performed.
Results
[DNA concentration fraction 1/ DNA concentration fraction 2] = 1.498 [RNA concentration fraction 2/ RNA concentration fraction 1] = 4.104
Example 2
A frozen cell pellet of 1 x 106 HL60 cultured cells was lysed in 100 I Solution A. The lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 500 μ\ Solution A were added and gently mixed. To this solution, 100 μ\ 96 %(v/v) ethanol were added. The final concentrations were:
13.7 % (v/v) ethanol 3.0 M guanidinium thiocyanate 21.5 mM trisodium citrate
Experiments and calculations were performed as described in Example 1 with the exception that in step (a) 6 mg of ferrimagnetic particles were suspended in 20 μ\ 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). Results
[DNA concentration fraction 1/ DNA concentration fraction 2] = 1.115 [RNA concentration fraction 2/ RNA concentration fraction 1] = 4.637
Example 3
A frozen cell pellet of 1 x 106 HL60 cultured cells was lysed in 100 μ\ Solution A. The lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 100 μ\ Solution A were added and gently mixed. To this solution, 500 μ\ 96 %(v/v) ethanol were added. The final concentrations were:
68.6 % (v/v) ethanol 1.0 M guanidinium thiocyanate 7.2 mM trisodium citrate
Experiments and calculations were performed as described in Example 1 with the exception that in step (a) 6 mg of ferrimagnetic particles were suspended in 20 μ\ 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).
Results
[DNA concentration fraction 1/ DNA concentration fraction 2] = 2.303 [RNA concentration fraction 2/ RNA concentration fraction 1] = 1.301 Example 4
A frozen cell pellet of 1 x 106 HL60 cultured cells was lysed in 100 /I Solution A. The lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 100 /I Solution A were added and gently mixed. To this solution, 500 μ\ 100 %(v/v) iso-propanol were added. The final concentrations were:
71.5 % (v/v) iso-propanol 1.0 M guanidinium thiocyanate 7.2 mM trisodium citrate
Experiments and calculations were performed as described in Example 1 with the exception that in step (a) 6 mg of ferrimagnetic particles were suspended in 20 μ\ 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).
Results
[DNA concentration fraction 1/ DNA concentration fraction 2] = 1.414 [RNA concentration fraction 2/ RNA concentration fraction 1] = 1.033
Example 5
Experimental setup
A frozen cell pellet of 1 x 106 HL60 cultured cells was lysed in 100 /I Solution B. The lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 300 μ\ Solution B were added and gently mixed. To this solution, 300 μ\ 96 %(v/v) ethanol were added. The final concentrations were:
41.1 % (v/v) ethanol
4.1 M guanidinium hydrochloride The subsequent isolation of nucleic acids comprises the steps of:
(a) 6 mg of ferrimagnetic particles according to WO 04/003231 were suspended in 20 μ\ of an aqueous solution comprising a composition according to the final composition of the lysate as displayed above
(41.1 %(v/v) ethanol, 4.1 M guanidinium hydrochloride). This suspension was added to the lysate, incubated for 10 seconds and the ferrimagnetic particles were removed from the lysate by a magnet (fraction 1).
(b) 60 μ\ 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.
(c) The fraction 1 and fraction 2 ferrimagnetic particles were given parallel identical treatments using the QIAGEN MagAttract RNA kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The optional DNase treatment was not performed in the case of both fractions of ferrimagnetic beads.
Real-time PCR ΔCt values
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 ΔCt. ΔCt is commonly used as a measure for RNA fraction of total nucleic acid. Larger ΔCt values identify higher RNA fractions of total nucleic acids. Results
ΔCt fraction 1 : 4.25 ΔCt fraction 2: 4.86
Example 6
A frozen cell pellet of 1 x 106 HL60 cultured cells was lysed in 100 μ\ Solution A. The lysate was further homogenized by 5 times passing a 23G syringe. Subsequently, 300 μ\ Solution A were added and gently mixed. To this solution, 300 μ\ 100 %(v/v) methanol were added. The final concentrations were:
42.8 % (v/v) methanol 2.0 M guanidinium thiocyanate 14.4 mM trisodium citrate
Experiments and calculations were performed as described in Example 5 with the exception that in step (a) 6 mg of ferrimagnetic particles were suspended in 20 μ\ 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).
Results
ΔCt fraction 1 : 4.72 ΔCt fraction 2: 5.94 Example 7
Experimental setup
A frozen cell pellet of 1 x 106 HL60 cultured cells was lysed in 100 μ\ Solution A. Subsequently, 100 μ\ Solution A were added and gently mixed. To this solution, 150 μ\ 96 %(v/v) ethanol were added. The final concentrations were:
41.1 % (v/v) ethanol 2.0 M guanidinium thiocyanate 14.4 mM trisodium citrate
The subsequent isolation of nucleic acids comprises the steps of:
(a) 0.6 mg of ferrimagnetic particles according to WO 04/003231 were suspended in 2 μ\ of an aqueous solution comprising a composition according to the final composition of the lysate as displayed above (41.1 %(v/v) ethanol, 2.0 M guanidinium thiocyanate, 14.4 mM trisodium citrate). This suspension was added to the lysate, incubated for 10 seconds and the ferrimagnetic particles were removed from the lysate by a magnet
(fraction 1).
(b) 80 μ\ 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.
Subsequently, the ferrimagnetic particles were removed from the lysate by a magnet (fraction 2) and the remaining solution was discarded.
(c) The fraction 1 and fraction 2 ferrimagnetic particles were given parallel identical treatments using the QIAGEN MagAttract RNA kit (QIAGEN, Hilden,
Germany) according to the manufacturer's instructions. The optional DNase treatment was not performed in the case of fraction 1 of ferrimagnetic beads, but the optional DNase treatment was performed in the case of fraction 2 of ferrimagnetic beads. Results
Nucleic acid yield in fraction 1 as determined from OD: 2.8 μg Nucleic acid yield in fraction 2 as determined from OD: 2.3 μg RNA/DNA ratio in fraction 1 determined from real-time PCR: 1.3 RNA/DNA ratio in fraction 2 determined from real-time PCR: 23.7

Claims

Claims
1. A method for sequentially isolating DNA and RNA from the same nucleic acid- containing sample, comprising the steps of:
(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),
(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,
(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,
wherein the time period of step (c) is less than half of the time period of step (d).
2. A method according to claim 1 , wherein the functional surface utilized in step (d) of claim 1 is of the same kind as the functional surface of step (c) of claim 1.
3. A method according to claim 1 or 2, wherein the time period of step (c) of claim 1 is less than 1/10 of the time period of step (d) of claim 1, preferably the time period of step (c) of claim 1 is less than 1/20 of the time period of step (d) of claim 1.
4. A method according to claim 1, 2 or 3, wherein the time period of step (c) of claim 1 is between 5 seconds and 60 seconds.
5. A method according to claim 1 , 2 or 3, wherein the time period of step (d) of claim 1 is 30 seconds or more.
6. A method according to claim 1 , wherein the chaotropic salt in step (a) of claim 1 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 a mixture thereof.
7. A method according to claim 1 or 6, wherein the final concentration of the chaotropic salt is in a range of from 1.2 to 3.5 M, preferably in a range of from 1.5 to 3 M.
8. A method according to claim 1 , 6 or 7, wherein the chaotropic salt in step (a) of claim 1 is added as a solid.
9. A method according to claim 1 , 6 or 7, wherein the chaotropic salt in step (a) of claim 1 is added as a solution.
10. A method according to claim 1 , wherein the alcohol in step (b) of claim 1 is selected from methanol, ethanol, n-propanol, iso-propanol or a mixture thereof.
11. A method according to claim 1 or 10, wherein the final concentration of the alcohol is in a range of from 25 to 60 %(v/v), preferably in a range of from 30 to 50 %(v/v).
12. A method according to claim 1 or 2, wherein the functional surface comprises carboxylic acid groups.
13. A method according to claim 1 or 2, wherein the functional surface is a silica surface.
14. A method according to claim 1, 2, 12, or 13, wherein the functional surface is provided in bead form, preferably the functional surface is a plurality of beads.
15. A method according to claim 14, wherein the beads are magnetic beads.
16. A method according to claim 14 or 15, wherein the number of beads utilized in step (c) of claim 1 is less than half of the number of beads utilized in step (d) of claim 1 , preferably the number of beads utilized in step (c) of claim 1 is less than
1/5 of the number of beads utilized in step (d) of claim 1 , more preferably the number of beads utilized in step (c) of claim 1 is less than 1/10 of the number of beads utilized in step (d) of claim 1.
17. A method according to claim 1 , wherein the DNA bound to the functional surface of step (c) of claim 1 after breaking the contact between the solution of step (b) of claim 1 and the functional surface is further purified.
18. A method according to claim 17, wherein the further purification of the DNA comprises at least the steps of:
(1 ) washing the DNA bound to the functional surface of step (c) of claim 1 after breaking the contact between the solution of step (b) of claim 1 and the functional surface with a suitable solution,
(2) eluting the DNA from the functional surface using a suitable solution,
(3) prior to, simultaneously with or following step (1) or step (2), performing a RNase treatment under suitable conditions.
19. A method according to claim 1 , wherein the RNA bound to the functional surface of step (d) of claim 1 after breaking the contact between the DNA-depleted solution of step (c) of claim 1 and the functional surface is further purified.
20. A method according to claim 19, wherein the further purification of the RNA comprises at least the steps of:
(1) washing the RNA bound to the functional surface of step (d) of claim 1 after breaking the contact between the DNA-depleted solution of step (c) of claim 1 and the functional surface with a suitable solution,
(2) eluting the RNA from the functional surface using a suitable solution,
(3) prior to, simultaneously with or following step (1) or step (2), performing a
DNase treatment under suitable conditions.
21. A method according to any of the claims 1 to 20, wherein the method is performed in an automated process.
22. A kit for performing a method according to any of the claims 1 to 21 , at least comprising:
(i) a functional surface, and/or
(ii) a chaotropic salt, and/or
(iii) an alcohol.
23. A kit according to claim 22 furthermore comprising:
(iv) a set of substances and/or devices to further purify the DNA obtained in step (c) of claim 1 and/or to further purify the RNA obtained in step (d) of claim 1.
EP04739565A 2003-06-04 2004-06-03 Method for sequentially isolating dna and rna from the same nucleic acid-containing sample Ceased EP1633869A1 (en)

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