Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
  • B01D15/363—Anion-exchange
• • 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
  • C12N15/101—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 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

• This invention relates generally to methods for separating nucleic acids such as genomic DNA, plasmid DNA and mRNA from contaminating cellular components such as proteins, lipids, soluble membrane components and the like.
• the invention relates to the improved recovery of nucleic acids from anion exchange chromatography media in either batch or packed mode by the addition of a composition that increases the pH of the elution solution.
• Genomic DNA isolated from blood, tissue or cultured cells has several applications, which include PCR, sequencing, genotyping, hybridization and southern blotting. Plasmid DNA has been utilized in sequencing, PCR, in the development of vaccines and in gene therapy. Isolated RNA has a variety of downstream applications, including blot hybridization, in vitro translation, cDNA synthesis and RT- PCR.
• the analysis and in vitro manipulation of nucleic acids is typically preceded by a nucleic acid isolation step in order to free the nucleic acid from unwanted contaminants which may interfere with subsequent processing procedures.
• nucleic acids are required as the first step.
• cells or homogenized tissue samples containing the nucleic acid of interest are harvested and lysed using standard methods, for example using enzymes such as Proteinase K and lysozyme; detergents, such as SDS, Brij, Triton XlOO, or using other chemicals such as sodium hydroxide, guanidium isothiocyanate, etc.
• enzymes such as Proteinase K and lysozyme
• detergents such as SDS, Brij, Triton XlOO
• other chemicals such as sodium hydroxide, guanidium isothiocyanate, etc.
• RNA may be removed or reduced if required by treatment of the enzymes such as RNAse.
• the presence of contaminants such as salts, phenol, detergents and the like can interfere with many downstream manipulations for which the nucleic acid is intended.
• Plasmid DNA, genomic DNA and RNA have similar charge properties to one another and are polyanions having high charge density. Binding to positively charged ion exchange resins is therefore possible in the presence of up to 0.7M sodium chloride, depending on the length and conformation of the nucleic acid to be adsorbed. An increase in nucleic acid length as well as double stranded conformation results in an increase in binding strength between the nucleic acid and the anion exchanger. However, this effect is only proportional to nucleic acid length up to about 2 kilobases.
• nucleic acids can be efficiently eluted from anion exchange resins under conditions of high salt concentration and the presence in the elution solution of an additive that increases the pH of the solution, the pH of the solution being suitably in the range of about pH 9 to about pH 13, and more preferably in the range of about pH 10 to about pH 12.
• an additive is guanidine or a guanidine-like derivative.
• Another example is a potassium carbonate. The addition of either additive to the elution solution has been shown to improve recovery of nucleic acids from anion exchange resins from 20-50% to 70-95%.
• the present invention provides a method for the separation and/or purification of a nucleic acid from cells, comprising: a) generating an aqueous solution containing the nucleic acid by lysing said cells with a lysis solution; b) contacting the aqueous solution containing the nucleic acid with an anion exchanger bound to a solid support matrix under conditions such that the anion exchanger binds the nucleic acid; and c) eluting said anion exchanger with an aqueous mobile phase comprising a nucleic acid elution salt solution; characterised in that the said elution solution comprises an additive such that the pH of the aqueous mobile phase is between about pH 9 and about pH 13, wherein the presence of the additive in the elution solution provides an increase in the nucleic acid recovery from the anion exchanger, as compared with the recovery of said nucleic acid in the absence of the additive.
• nucleic acids consist of a chain (or a paired chain of deoxyribose phosphate monomers covalently linked via phosphodiester bonds, each sugar phosphate moiety carrying a single aromatic heterocyclic base: adenine (A), guanine (G), cytosine (C), thymine (T found solely in DNA), and uracil (U found solely in RNA).
• DNA is thus a polyanion, where the net negative charge of the nucleic acid molecule is related directly to chain length.
• Nucleic acids therefore, display strong binding affinities to anion exchange resins such that a high salt concentration in the elution solution is required to efficiently remove the nucleic acid from the resin.
• guanidine and guanidine-like compounds particularly arginine on recovery of nucleic acids from anion exchange resins is most pronounced at alkaline pH, for example, between about pH 9 and about pH 13, more particularly at pH values between about 10 and 12.
• a pH range of between about 10.5 and 11.6 appears to provide optimum recovery.
• Recovery of nucleic acids is highest when arginine or guanidine carbonate is added to the elution solution.
• one of the unique properties of the guanidinium group is its delocalized positive charge property which is due to the conjugation between the double bond and the nitrogen lone pairs.
• the guanidinium group is able to form multiple hydrogen-bonds preferentially with guanine bases, which may act to cause local deformation of the nucleic acid structure, which change in conformation of nucleic acids contributes to a change in desorption kinetics thereby favouring high recoveries of nucleic acids during anion exchange chromatography.
• Macromolecules are known to bind to adsorptive surfaces through multiple points of attachment creating microenvironments on chromatography media surfaces, which allow adsorption that can become close to irreversible with conventional desorption techniques.
• the additive is a compound having the formula (I)
• R is selected from H, and lower alkyl, optionally substituted by amino.
• lower alkyl is a Ci to C 4 alkyl group, for example methyl, ethyl, propyl and butyl, preferably, methyl or ethyl.
• R is an amino-substituted lower alkyl group
• examples of the compounds according to formula (I) include 2-aminoethyl-guanidine, 3-aminopropyl-guanidine and 4-aminobutyl-guanidine (agmatine).
• R is hydrogen, thus compound (I) is guanidine, as its carbonate or bicarbonate salt.
• the additive is a compound having the formula (I):
• the additive is arginine, suitably L-arginine, D-arginine, or a mixture of both optical isomers.
• the additive is an inorganic salt that provides a similar pH.
• a salt is potassium carbonate.
• Another example is sodium carbonate.
• the guanidine and guanidine-like compound or the potassium carbonate is present as an additive in the elution solution at a concentration of between 0.1 and 2 Molar, preferably between 0.25M and 0.5M.
• the elution solution will typically comprise a salt solution, suitably between
• the pH of the aqueous mobile phase is between about pH 9 and about pH 13, the preferred range of pH being between about pH 10 and about pH 12, more preferably between about pH 10.5 and about
• nucleic acid refers to any DNA or RNA molecule, or a
• DNA/RNA hybrid DNA/RNA hybrid, or mixtures of DNA and/or RNA.
• nucleic acid therefore is intended to include genomic or chromosomal DNA, plasmid DNA, amplified DNA, total RNA and mRNA.
• the process according to the present invention is particularly suitable for the preparation and/or purification of genomic DNA derived from complex mixtures of components derived from cellular and tissue samples from any recognised source, including normal and transformed cells, with respect to species (e.g. human, rodent, simian), tissue source (e.g. brain, liver, lung, heart, kidney skin, muscle) and cell type (e.g. epithelial, endothelial, blood).
• species e.g. human, rodent, simian
• tissue source e.g. brain, liver, lung, heart, kidney skin, muscle
• cell type e.g. epithelial, endothelial, blood
• the present method is suitable for the preparation and/or purification of genomic DNA having a size of from about 0.1 kilo-bases to about 200 kilo-bases, or of plasmid DNA, cosmid, BAC or YAC.
• the present invention is useful for purifying plasmid DNA and cosmid DNA, in particular for downstream applications in molecular biological research, such as cloning and sequencing, gene therapy and in diagnostic applications both in vivo and in vitro.
• Anion exchange resins suitable for use with methods of the present invention include both strong anion exchangers and weak anion exchangers, wherein the anion exchange resin suitably comprises a support carrier to which charged or chargable groups have been attached.
• the ion exchange resin may take the form of a bead, a membrane or a surface.
• strong anion exchange resins include Q-sepharose fast flow resin, Q-sepharose XL and CaptoQ.
• Examples of weak ion exchange resins include ANX fast flow resin and DEAE Sephadex A25 resin. (GE Healthcare)
• an additive of disclosed above in the aqueous mobile phase it is possible to increase recovery of nucleic acid from the anion exchanger of at least 40% and typically between about 40% and about 400%, as compared with the recovery of said nucleic acid from the same anion exchanger and in the absence of the additive in the elution solution, all other conditions being equal.
• the invention provides a kit for the separation and/or purification of nucleic acid from a cellular sample, the kit comprising a lysis solution for generating an aqueous solution containing the nucleic acid from the cellular sample; an anion exchanger bound to a solid support matrix for binding the nucleic acid; an elution solution for eluting the nucleic acid from the anion exchanger; and optionally desalting means for desalting the eluted nucleic acid.
• a lysis solution for generating an aqueous solution containing the nucleic acid from the cellular sample
• an anion exchanger bound to a solid support matrix for binding the nucleic acid
• an elution solution for eluting the nucleic acid from the anion exchanger
• optionally desalting means for desalting the eluted nucleic acid.
• an additive such that the pH of said elution solution is between about pH 9 and about pH 13.
• the additive is arginine. In another embodiment the additive is guanidine present as its carbonate or bicarbonate salt. In yet another embodiment, the additive is potassium carbonate.
• the anion exchanger is ANX fast flow resin.
• the anion exchanger is DEAE Sephadex A25 resin, Q-sepharose fast flow resin, Q-sepharose XL or CaptoQ resin (all from GE Healthcare).
• Figure 1 shows pulse-field gel electrophoresis analysis of purified genomic DNA from human blood samples, using a method according to one embodiment of the invention.
• Figure 2 shows an agarose gel image of purified genomic DNA from human blood samples.
• Figure 3 shows comparison of restriction enzyme (EcoRI) digested and un-digested genomic DNA that was purified from human blood samples.
• Figure 4 shows real time PCR amplification results obtained from the genomic DNA samples from human blood, with very similar amplification profiles observed among the samples.
• EcoRI restriction enzyme
• Figure 5 shows pulse-field gel electrophoresis analysis of purified genomic DNA from rat liver samples, using a method according to one embodiment of the invention.
• Figure 6 shows real time PCR amplification results obtained from the genomic DNA samples from rat liver samples, with very similar amplification profiles observed among the samples.
• Figure 7 shows comparison of restriction enzyme (Hindlll) digested and un-digested genomic DNA that was purified from rat liver samples.
• Figure 8 shows pulse-field gel electrophoresis analysis of purified genomic DNA from cultured MRC5 cells samples, using a method according to one embodiment of the invention.
• Figure 9 shows comparison of restriction enzyme (EcoRI) digested and un-digested genomic DNA that was purified from MRC5 cells samples.
• Genomic DNA isolation from blood is done in 2 steps.
• the first step is the lysis of blood and the second step is purification of genomic DNA using ion-exchange column chromatography.
• Lysis This process involved 2 steps. First white blood cells are isolated and then the isolated white blood cells are lysed using lysis solution.
• the protocol used for the isolation of white blood cells and lysis of white blood cells is as follows. Five ml of blood is used as an example here. However, the protocol can be adjusted accordingly based on the amount of blood used.
• the purification process also includes a de-salting process (steps 17-21).
• Desalting 17. Remove the cap of desalting column and discard the solution. Cut the closed end of the column at notch and place the column in a centrifuge tube using the adaptor.
• the tissue sample is prepared by the following steps. It is critical to have a completely homogenized sample to obtain good yield of genomic DNA from the purification process.
• Tissue samples so prepared are subjected to the following steps for the isolation of genomic DNA. Steps 4-7 are for sample lysis; steps 8-12 are for purification; and steps 13-17 are for de-salting.
• RNAse A solution (20 mg/ml) and incubate at 37 0 C for 15 min. 7. Dilute the crude lysate with 4ml of DNAse free water and 5ml of Loading solution and centrifuge at 5000xg for 15 min to pellet particulates.
• Purification 8 Remove the cap from the top of purification column. Discard the solution by decanting. Cut the closed end of the column at the notch and place the columns in 50ml centrifuge tubes using column adaptors.
• Quantitation of the purified genomic DNA samples was achieved with a UV spectrophotometer, using 1 x TE Buffer pH8.0 as the blank and lcm path length cuvettes.
• Lysisl solution 30 rnM Tris-HCl, 10 mM Magnesium chloride, 2% Triton X 100 and 0.6 M sucrose.
• Lysis2 solution 20 mM Tris-HCl, 20 mM EDTA, 20 mM sodium chloride and
• Loading solution 700 mM sodium chloride,50 mM Tris and ImM EDTA.
• Tissue protocol Lysis solution: 20 mM Tris-HCl, 20 mM EDTA, 100 mM sodium chloride and
• Loading solution 700 mM sodium chloride, 50 mM Tris and ImM EDTA.
• ANX Sepharose fast flow (high sub) resins were used here. These resins are very stable over a wide pH range (3-13), with an average particle size of 90 ⁇ m (We have subsequently tested other anion exchange resins and found they work well too).
• the columns were pre-packed using a salt solution having similar strength as the sample loading solution, with suitable anti-microbial agent (e.g. ethanol or kethon). This eliminates the need for column equilibration prior to loading of the sample in loading solution for binding of the nucleic acid.
• suitable anti-microbial agent e.g. ethanol or kethon
• the source cell used here was human blood and the blood protocol described in (A) (a) above was followed. We found that even when a salt concentration of 2 to 3M was used it did not significantly improve the recovery of genomic DNA from the ion- exchange columns. After elution all the samples were desalted using NAP-IO or NAP-25 columns (GE Healthcare) and the DNA was quantified using UV spectrophotometer. To identify a buffer or a solution which could provide better recovery, different combinations of salts were evaluated.
• sodium hydroxide is not only caustic, but may also lead to irreversible denaturation of nucleic acids and degradation over time.
• pH appears to play a critical role in the recovery of nucleic acid in addition to the salt strength.
• Combination of 1 to 2 M sodium chloride, with 0.25 to 0.5M arginine, 0.5M potassium carbonate or 0.5M guanidine carbonate can be used for improved elution.
• 0.5 to 1 M sodium carbonate or 0.5M to 1 M Tris base can also be used to increase the elution as well.
• the quality of the purified genomic DNA was assessed by several methods.
• the DNA was subjected to restriction enzyme digest using EcoRI.
• Purified genomic DNA 250 ug was digested with 40 units of the enzyme.
• the digested sample was analyzed on an agarose gel side-by-side with un-digested sample DNA. The gel image shows that all the genomic DNA was completely digested ( Figure 3, Lanes 2, 4, 6 represent the purified, un-digested genomic DNA, while Lanes 1, 3, 5 are samples digested with the enzyme).
• the quality of the genomic DNA samples was indirectly measured by the efficiency in a multiplex PCR reaction.
• a long range multiplex PCR for the P450 genes were used for this test (CodeLink P450 protocol, GE Healthcare).
• Three amplicons from genes CYP2D6, CYP3A4, and CYP3A5 were amplified in a single reaction.
• the size in amplicons ranges from 335 bp to 2600 bp.
• the size and yield of the PCR products were determined via the Agilent Biolanalyzer 2100 and DNA 7500 kit.
• the multiplex PCR reactions worked well for all the samples tested (data not shown).
• the quality of the genomic DNA samples was also tested by real time PCR assays. Real time PCR experiments were done using Applied Biosystems 7900HT Fast
• the quality of the purified genomic DNA was assessed by several methods.
• the quality of the genomic DNA samples was tested by real time PCR assays. Real time PCR experiments were performed using Applied Biosystems 7900HT Fast Real Time PCR System. All the samples tested show very similar amplication profiles (Figure 6).
• the DNA was subjected to restriction enzyme digest using HindIIL Purified genomic DNA (250 ug) was digested with 40 units of the enzyme.
• the digested sample was analyzed on an agarose gel side-by-side with un-digested sample DNA.
• the gel image shows that all the genomic DNA was completely digested ( Figure 7, Lanes 2, 4, 6, 8 represent the purified, un-digested genomic DNA, while Lanes 1, 3, 5, 7 are samples digested with the enzyme).
• the crude lysate was diluted with loading solution and transferred to the ion-exchange purification column. After all the solution passed through the resin, 5ml of loading solution was added to the column. When there was no more solution left on the top of the resin, 2.5ml of elution solution was added to the column and the product was collected in the eluate.
• the genomic DNA thus obtained was desalted using NAP -25 column. The purity of the product was assessed by UV spectrophotometry and by gel analysis. The size of the genomic DNA isolated was determined by Pulse-Field Gel Electrophoresis. The genomic DNA obtained by this method was also evaluated in downstream applications such as real time PCR and restriction digestion.
• the quality of the purified genomic DNA was assessed by restriction digest.
• the DNA was subjected to restriction enzyme digest using EcoRI.
• Purified genomic DNA 250ug was digested with 40 units of the enzyme.
• the digested sample was analyzed on an agarose gel side-by-side with un-digested sample DNA.
• the gel image shows that all the genomic DNA was completely digested ( Figure 9, Lanes 2, 4, 6, 8, 10, 12 represent the purified, un-digested genomic DNA, while Lanes 1, 3, 5, 7, 9, 11 are samples digested with the enzyme).

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