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WO2008035991A2 - Procédé d'extraction d'acide nucléique - Google Patents

Procédé d'extraction d'acide nucléique Download PDF

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Publication number
WO2008035991A2
WO2008035991A2 PCT/NZ2007/000273 NZ2007000273W WO2008035991A2 WO 2008035991 A2 WO2008035991 A2 WO 2008035991A2 NZ 2007000273 W NZ2007000273 W NZ 2007000273W WO 2008035991 A2 WO2008035991 A2 WO 2008035991A2
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WIPO (PCT)
Prior art keywords
nucleic acid
solution
biological material
dna
tissue
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PCT/NZ2007/000273
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English (en)
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WO2008035991A3 (fr
Inventor
Michael Ronald Cook
Original Assignee
Michael Ronald Cook
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Publication of WO2008035991A2 publication Critical patent/WO2008035991A2/fr
Publication of WO2008035991A3 publication Critical patent/WO2008035991A3/fr

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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
    • 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

  • nucleic acids from biological material are known in the art. Many procedures involve binding the nucleic acid to a matrix having affinity for the nucleic acid, to be isolated, under certain conditions. The matrix-bound nucleic acid may then be washed to remove unwanted material, before the nucleic acid is eluted from the matrix under different conditions in which the matrix has no affinity for the nucleic acid. In one example of such a method (WO 99/29703), negatively charged nucleic acid is bound at a relatively acidic pH, washed, then eluted at a relatively basic pH.
  • the invention provides a method of isolating nucleic acid from biological material, the method comprising the steps: a) providing biological material suspended in a solution, b) adjusting the pH of the suspension from a) to within the range about pH 1 to about pH 6, c) contacting nucleic acid in the suspension from b) with an affinity matrix capable of binding the nucleic acid in the pH range about pH 1 to about pH 6, in order to bind the nucleic acid to the affinity matrix; and d) eluting the nucleic acid from the affinity matrix using a buffer with a pH of more than 8, wherein the method does not require a centrifugation or filtration step prior to step c).
  • the biological material is selected from a group consisting of animal tissue, plant tissue, animal cells, mammalian cells, plant cells, bacterial cells, yeast cells and fungal cells.
  • the biological material is the biological material is plant tissue.
  • the plant tissue is freeze-dried prior to suspension in the solution of step a).
  • the biological material is plant leaf tissue.
  • the solution in a) comprises a detergent.
  • the solution in a) comprises a nuclease.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA.
  • all steps are performed in the wells of multi-well plates.
  • nucleic acid is eluted from the affinity matrix into a solution with a pH of more than 8, more preferably more than 9.
  • the invention provides a method of isolating nucleic acid from biological material, the method comprising the steps: a) providing biological material suspended in a solution, b) adjusting the pH of the suspension from a) to within the range about pH 1 to about pH 6, c) contacting nucleic acid in the suspension from b) with an affinity matrix capable of binding the nucleic acid in the pH range about pH 1 to about pH 6, in order to bind the nucleic acid to the affinity matrix; and d) eluting the nucleic acid from the affinity using a buffer with a pH of more than 8, wherein the method does not require a centrifugation or filtration step prior to step c).
  • nucleic acid means separating the nucleic acid from its normal cellular environment, to substantially purify the nucleic acid.
  • the "isolated" nucleic acid is greater than 50% pure, more preferably greater than 60% pure, more preferably greater than 70% pure, more preferably greater than 80% pure, more preferably greater than 90% pure and most preferably greater than 95% pure.
  • filter or grammatical equivalents thereof as used herein refers to common usage of the term, that is passing a solution or suspension through a filter, strainer, sieve, membrane or layer to separate particulate matter or tissue or cellular debris from the liquid phase.
  • filter does not include aspiration of the liquid phase from a suspension through a pipette or pipette tip avoiding particulate matter or tissue or cellular debris in the suspension.
  • the biological- material from which nucleic acid is to be purified may be of any type and may be selected from a group consisting of animal tissue, plant tissue, fungal tissue, animal cells, mammalian cells, plant cells, bacterial cells, yeast cells and fungal cells.
  • the biological material is plant tissue. More preferably the plant tissue is leaf tissue.
  • the plant tissue is freeze-dried. Methods for freeze-drying plant tissue are known to those skilled in the art. Any suitable freeze-drying method may be used. For example the tissue may be freeze-dried at ⁇ lOOmTorr at approximately -95°C for 24 hours.
  • the biological material is ground prior to being suspending in the solution of step a).
  • This grounding may be achieved by any suitable method.
  • a preferred method of grinding is by use of a mill, such as the A-Tech (New Zealand) Tissue Disintegrator, or similar apparatus.
  • the nucleic acid to be isolated is DNA.
  • the DNA may be selected from but is not limited to genomic DNA, plasmid DNA, mitochondrial DNA or plastid DNA.
  • the DNA is genomic DNA.
  • the nucleic acid to be isolated is RNA. 4. The solution in step a)
  • the pH of the solution in step a) is in the range pH 1.0 to pH 12.0, more preferably pH 3.0 to pH 1 1.0, more preferably pH 5.0 to pH 10.0, more preferably pH 7.0 to pH 9.0, more preferably pH 8.0 to pH 9.0. Most preferably the pH of the solution in step a) is about 8.5.
  • the solution in step a) is buffered.
  • Any suitable buffer may be used.
  • Preferred buffers include Tris-HCl, MES and NaOAc.
  • a preferred buffer is MES.
  • MES is included at a concentration in the range about 25mM to about 50OmM, more preferably in the range about 5OmM to about 40OmM, more preferably in the range about 75mM to about 30OmM, more preferably in the range about 10OmM to about 20OmM.
  • MES is included at about 15OmM.
  • the nuclease when the nucleic acid to be isolated is DNA the nuclease, optionally included, in the solution of a) is preferably an RNAse, preferably RNAse A.
  • the RNAse A is at a concentration of in the range 1 to 100ug/ml, more preferably 2 to 90ug/ml, more preferably 3 to 80ug/ml, more preferably 4 to 70ug/ml, more preferably 5 to 60ug/ml, more preferably 6 to 50ug/ml, more preferably 7 to 40ug/ml, more preferably 8 to 30ug/ml, more preferably 9 to 20ug/ml, most preferably at lOug/ml.
  • the nuclease, optionally included, in the solution in step a) is preferably a DNAse. Any suitable DNAse may be used.
  • a suitable DNAse is RQl RNA-free DNAse (Promega, USA, Cat No. 610a).
  • Preferably the DNAse is used at a concentration of about 50u/ml.
  • the detergent in the solution in step a) may be anionic, non-ionic, cationic or zwitterionic or a mixture.
  • the detergent is an anionic detergent, for example an alkyl sulphate or alkyl sulphonate.
  • the detergent in the solution in a is SDS.
  • the SDS is at a concentration between 0.1 and 5%, more preferably between 0.2 and 0.8%; more preferably between 0.3 and 0.7%, more preferably between 0.4 and 0.6%.
  • Most preferably the SDS is at a concentration of 0.5% w/v.
  • the detergent in the solution in a is Triton X-100.
  • the Triton X- 100 is at a concentration between 0.1 and 5%, more preferably between 0.2 and 0.8%; more preferably between 0.3 and 0.7%, more preferably between 0.4 and 0.6%.
  • the Triton X- 100 is at a concentration of 0.5% w/v.
  • the solution in step a) may include two detergents.
  • a combination of SDS and Triton X-100 is preferred.
  • Suitable detergents include but are not limited to CTAB, Sodium deoxycholate and sarkosyl.
  • the solution in step a) contains a chaotropic agent such as, for example, guanidine hydrochloride or urea.
  • a chaotropic agent such as, for example, guanidine hydrochloride or urea.
  • Guanidine hydrochloride may be used at a concentration in the range 6 to 8M.
  • the chaotropic agent is urea is used at a concentration in the range 0.1 to 10.0M, more preferably 0.1 to 1.0M.
  • the solution of step a) preferably includes an antioxidant.
  • an antioxidant may be used including for example ⁇ -mercaptoethanol, dithiothreitol (DTT) or sodium metabisulphate
  • SMBS ⁇ -mercaptoethanol
  • DTT DTT
  • ⁇ -ME included at a concentration in the range about 0.05% to about 0.5%, more preferably about 0.06% to about 0.4%, more preferably about 0.07% to about 0.3%, more preferably about 0.08% to about 0.2%, more preferably about 0.09% to about 0.15%. Most preferably ⁇ -ME is included at a concentration of about 0.1% w/v.
  • DTT is included at a concentration in the range from about 1 OmM to about 10OmM, more preferably 2OmM to about 8OmM, more preferably 3OmM to about 7OmM, more preferably 4OmM to about 6OmM. Most preferably DTT is included at a concentration of about 5OmM.
  • an ion-chelating agent is included in the solution of step a). Any suitable ion-chelating agent may be used.
  • a preferred ion-chelating agent is EDTA.
  • EDTA is included at a concentration in the range about 5mM to about 5OmM, more preferably about 1OmM to about 4OmM, more preferably about 15mM to about 35mM, more preferably about 2OmM to about 3OmM.
  • EDTA is included at a concentration of about 25mM.
  • a polyphenol absorber is included in the solution of step a). Any suitable polyphenol absorber may be used.
  • a preferred polyphenol absorber is polyvinyl pyrrolidone.
  • polyvinyl pyrrolidone is included at a concentration in the range about 1% to about 4%, more preferably 1% to about 3%. Most preferably the polyvinyl pyrrolidone is included at a concentration of about 2%.
  • the solution in step a) preferably contains one or more components selected from proteases, salts and solvents.
  • biological material is agitated to encourage formation of the homogeneous suspension. This may be achieved in numerous ways, for example by gentle vortexing, drawing the buffer and biological material in and out of a pipette or pipette tip, or by use of a shaker, such as a plate shaker.
  • Adjustment of pH in step b) may be effected by adding an acidic solution or buffer to the suspension of step a). Any compatible solution or buffer may be used. Acidity in the solution or buffer may be provided by HCl.
  • the buffer may be of the same composition as the solution in step a) but may also contain other components such as HCl to effect adjustment of the pH in step b) to within the range of pH 1 to pH 6.5. Preferably pH is adjusted to within the range 2 to 6.5, more preferably 3 to 6, more preferably 4 to 6, more preferably 4.5 to 5.5.
  • An alternative buffer for effecting pH adjustment is MES.
  • the MES has a pH of 5.2.
  • the MES is at a concentration of 200 mM, more preferably 10OmM, more preferably 50 mM and most preferably 25mM.
  • the solution in step a) should preferably include at least one of Polyvinyl Pyrrolidone (PVP) and Sodium MetaBisulphite (SMB).
  • PVP Polyvinyl Pyrrolidone
  • SMB Sodium MetaBisulphite
  • both PVP and SMB should be included.
  • both PVP and SMB should be included at 2% W/V.
  • affinity matrix Any suitable affinity matrix may be used.
  • a suitable affinity matrix for use in the invention is described in WO 99/29703.
  • a suitable solid phase is also marketed as ChargeSwitch by Invitrogen Life Technologies (USA).
  • the affinity matrix may incorporate histidine or a polyhistidine which will tend to bind nucleic acids at low pH e.g. less than 6 and will then release the bound nucleic acids when the pH is increased e.g. to greater than 8.
  • the nucleic acids are bound at substantially neutral pH to an aminated surface and released at very high pH.
  • the affinity matrix may incorporate ion exchange resins that are positively charged , where the charge can be reversed or made neutral by increasing the pH above its pKa.
  • ion exchange resins that are positively charged , where the charge can be reversed or made neutral by increasing the pH above its pKa.
  • nucleotides, polyamines, imidazole groups and other similar reagents with a suitable pKa value e.g. nucleotides, polyamines, imidazole groups and other similar reagents with a suitable pKa value.
  • the affinity matrix may be in the form of beads.
  • the beads may be magnetic to facilitate washing of the beads to remove unwanted material.
  • step b) and step c) are performed simultaneously.
  • the affinity matrix may be provided in a low pH buffer so that addition of the affinity matrix to the suspension from step a) adjusts the pH of the suspension to within the range pH 1 to pH 6.
  • the method may include a washing step between steps c) and d) to remove unwanted material from the affinity matrix prior to elution of the nucleic acid.
  • washing of the affinity matrix is with a solution, optionally buffered, with approximately neutral pH.
  • the wash solution has a pH in the range 6.1 to 7.9, more preferably a pH within the range 6.2 to 7.8, more preferably 6.3 to 7.7, more preferably 6.4 to 7.6, more preferably 6.5 to 7.5, more preferably 6.6 to 7.4, more preferably 6.7 to 7.3, more preferably 6.8 to 7.2, more preferably 6.9 to 7.1, most preferably a pH of 7.0.
  • One suitable wash solution for use in the method is water. 10. Elution of the nucleic acid
  • the buffer in step d) into which the nucleic acid is eluted has a pH in the range between pH 8 and pH 10.
  • a buffer is used that will not adversely effect downstream applications of the nucleic acid, such as PCR amplification.
  • the buffer is a Tris-HCl buffer, more preferably 10 mM Tris-HCl.
  • the buffer also contains EDTA, preferably at a concentration of 1 mM.
  • the method is carried out in multi-well plates.
  • Any suitable multi-well plate may be used including 6, 12, 24, 48, 96, 384 well plates.
  • Any multiwell plates could be used, preferably 96-well 1.2ml deepwell polypropelene plates are used.
  • the method is carried out on an automated liquid handling system.
  • the method could be split into manual and automated procedures and carried out on platforms such as Beckman Coulter's Biomek 2000, preferably the method would be fully automated as per ATech (NZ) 's DNA extraction system.
  • the advantages provided by the present method include removal of the precipitation/ centrifugation/filtration steps required prior to binding of the nucleic acid to the solid matrix in other methods.
  • the method of the invention is amenable to high throughput processing of multiple samples on any standard liquid handling workstation.
  • the method of the invention is used in a high-throughput multi-well plate-based set up, there is no need for use of an automated plate centrifuge or a vacuum/pressure filtration manifold. This is a significant advantage as automated plate centrifuges are expensive, and vacuum/pressure filtration manifolds can be unreliable.
  • the method of the invention allow the user to take use plates of tissue (optionally freeze-dried) and to grind the tissue in batches in a mill (such as the A-Tech (NZ) Tissue Disintegrator).
  • the plates may then me placed directly onto a liquid handler for complete automation through to purified DNA.
  • the plates can be used on a nucleic acid extraction system that incorporates an automated mill (such as the DNA Extraction System provided by A-Tech CNZ)), and experience autonomous extractions from intact leaf through to purified nucleic acid.
  • an automated mill such as the DNA Extraction System provided by A-Tech CNZ
  • Methods and kits currently available typically require the user to grind the biological material (e.g. plant leaf material), to add a lysis buffer, and then to centrifuge or filter the samples, to collect the supernatants which can then be loaded onto an automated workstation for completion of the final nucleic acid extraction steps.
  • biological material e.g. plant leaf material
  • the present invention removes the need for such centrifugation or filtration steps to separate unwanted biological material from the nucleic acid containing buffer prior to binding of the nucleic acid to the affinity matrix.
  • This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • Figure 1 shows an ethidium bromide stained agarose gel containing duplicate samples of DNA isolated by: the ChargeSwitch (Invitrogen Life Technologies, USA) protocol (Row 1), the “manual” protocol of method of the invention (Row 2), and the “automated” protocol of method of the invention (Row 3).
  • the ChargeSwitch Invitrogen Life Technologies, USA
  • Figure 2 shows an ethidium bromide stained agarose gel containing RAPD PCR products amplified from DNA isolated: by the "automated" protocol of method of the invention (lanes 1- 8) and a standard DNA extraction protocol (lanes Cl and C2). Lane L contains a DNA size standard.
  • Figure 3 shows an ethidium bromide stained agarose gel containing DNA samples isolated by the protocol of the method of the invention with alternative buffers and without PVP or SMB as described in Example 4.
  • Figure 4 shows an ethidium bromide stained agarose gel containing DNA samples isolated by the protocol of the method of the invention with alternative buffers and including PVP or SMB as described in Example 4.
  • Figure 5 shows ethidium bromide stained agarose gels containing RAPD PCR products amplified from DNA isolated by the "automated" protocol of method of the invention with alternative buffers and without PVP or SMB as described in Example 4.
  • Figure 6 shows ethidium bromide stained agarose gels containing RAPD PCR products amplified from DNA isolated by the "automated" protocol of method of the invention with alternative buffers and including PVP or SMB as described in Example 4.
  • Figure 7 shows an ethidium bromide stained agarose gel with quantified DNA from apple leaf tissue isolated using the method of the invention with variation in HCl added to the lysis buffer.
  • Figure 8 shows an ethidium bromide stained agarose gel with quantified DNA from apple leaf tissue isolated using the method of the invention with variation in PVP or SM added.
  • Figure 9 shows an ethidium bromide stained agarose gel with SNP marker analysis of 8 DNA samples extracted from kiwifruit leaf tissue using protocol 43. Note poor quality of lanes 1 and
  • Figure 10 shows an ethidium bromide stained agarose gel with SNP marker analysis of 8 DNA samples extracted from kiwifruit leaf tissue using protocol 182 (as in protocol 43, but increased NaCl in LBl and LB2 from 25OmM to 50OmM. Note poor quality of lanes 7 and 8.
  • Figure 1 1 shows an ethidium bromide stained agarose gel with SNP marker analysis of 8 DNA samples extracted from kiwifruit leaf tissue using protocol 183 (as in protocol 43, but increased NaCl in LBl and LB2 from 25OmM to 100OmM. Note poor quality of lane 4.
  • Figure 12 shows an ethidium bromide stained agarose gel with SNP marker analysis of 16 DNA samples extracted from kiwifruit leaf tissue using protocol 69 (as in protocol 43, but both LB 1 and LB2 contain 5OmM Dithiothreitol (DTT).
  • DTT Dithiothreitol
  • Figure 13 shows an ethidium bromide stained agarose gel with SNP marker analysis of 17 DNA samples extracted from kiwifruit leaf tissue using protocol 44 (as in protocol 43, but both LB land LB2 contain 25mM ⁇ -mercaptoethanol (BME).
  • Example 1 Isolation of nucleic acid by a "manual" protocol of the method of the invention using pH-based reversible binding of nucleic acid
  • Apple leaf material was harvested directly into 1.2ml x 96 deep-well plates, with approximately 20-30mg of fresh tissue in each well. The entire plate was freeze dried at ⁇ lOOmTorr (approx. - 95 0 C cold trap) for 24 hours. Two stainless steel balls of 4.76mm diameter were placed in each well, and the plate was sealed using EasyPierce plates seals (Cat. # AB-0757, ABgene (UK)), applied with the ABgene thermal plate sealer. The freeze-dried tissue was then stored at -20 0 C with desiccated silica until required.
  • EasyPierce plates seals Cat. # AB-0757, ABgene (UK)
  • LBl Lysis Buffer 1
  • microtitre plate Place the microtitre plate on a 24-pin magnetic separator (e.g. Invitrogen catalogue # CS 15096, or similar), and leave for 90 seconds to allow magnetic beads to form a pellet on the side of the wells. Note, if solution is viscous, longer incubations may be required. 9. Aspirate all liquid from the wells, ensuring the pellet remains intact.
  • a 24-pin magnetic separator e.g. Invitrogen catalogue # CS 15096, or similar
  • step 8 and 9 Resuspend pelleted beads with 150 ⁇ l of 1OmM Tris-HCl (pH 8.5), ImM EDTA (pH 8.0), or Invitrogen elution buffer
  • Example 2 Isolation of nucleic acid by an automated protocol
  • Lysis Buffer 1 (LBl) for all samples by combining the following volumes for each sample: 125 ⁇ l Invitrogen buffer L 18, 25 ⁇ l 10% SDS and 2 ⁇ l Invitrogen RNase (or similar).
  • microtitre plate placed on a 24-pin magnetic separator (e.g. Invitrogen catalogue # CS 15096, or similar), and leave for 90 seconds to allow magnetic beads to form a pellet on the side of the wells. Note, if solution is viscous, longer incubations may be required.
  • a 24-pin magnetic separator e.g. Invitrogen catalogue # CS 15096, or similar
  • step 10 Resuspend pelleted beads with 150 ⁇ l of 1OmM Tris-HCl (pH 8.5), ImM EDTA (pH 8.0), or Invitrogen elution buffer
  • Example 3 Optimisation of buffers in the method of the invention for use on a robot
  • Kiwifruit leaf material was prepared as described for apple leaf material in Example 1.
  • microtitre plate Place the microtitre plate on a 24-pin magnetic separator (e.g. Invitrogen catalogue # CS 15096, or similar), and leave for 90 seconds to allow magnetic beads to form a pellet on the side of the wells. Note, if solution is viscous, longer incubations may be required.
  • a 24-pin magnetic separator e.g. Invitrogen catalogue # CS 15096, or similar
  • Lysis Buffer 1 (LBl) was prepared by combining the following volumes for each sample: 125ul Invitrogen buffer L 18, 25ul 10% SDS and 2ul Invitrogen RNase (or similar).
  • Lysis Buffer 2 (LB2) was prepared by combining the following volumes for each sample: 125ul Invitrogen buffer Ll 8, l-5ul concentrated HCl was optionally added.
  • Results are shown in Figures 7 and 8 for HCl addition and PVP and SM addition respectively.
  • Example 4 Validation of the usefulness of the nucleic acid isolated by the method of the invention in downstream applications
  • Example 2 To validate the quality of the nucleic acid preparation produced and usefulness in downstream applications, the nucleic acid isolated in Example 2 was used as a template for PCR amplification using RAPD primer 5'-GGTCGGGTCA-S' under standard PCR conditions.
  • Lanes 1 -8 include PCR products amplified from replicate DNA samples produced by the method of example 2.
  • Lane L is a molecular weight under lanes Cl and C2 are the PCR products amplified from the control DNA extraction protocol.
  • the results show that RAPD fragments have been reliably amplified in all replicates 1- 8 in a manner comparable to those amplified from the control DNA samples in lane Cl and C2.
  • Apple leaf material was prepared as described in Example 1.
  • MES pH 5.2 was trialled at a range of concentrations ((25mM, 5OmM, 10OmM and 20OmM).
  • the protocol of Example 2 was also performed with or without 2% w/v Polyvinyl Pyrrolidone (PVP) and 2% w/v Sodium MetaBisulphate (SMB) in buffer LBl .
  • PVP Polyvinyl Pyrrolidone
  • SMB Sodium MetaBisulphate
  • PVP is able to bind phenolic groups and the applicant postulated the inclusion of PVP may improve yield and quality.
  • SMB is a reducing agent and was added to prevent oxidative degradation of the nucleic acid.
  • Results show that there is no significant difference in PCR performance over the MES concentration ranges.
  • Use 25mM MES pH 5.2 may be beneficial in order to reduce residual carryover of MES following washing.
  • Apple leaf material was prepared as described in Example 1.
  • microtitre plate Place the microtitre plate on a 24-pin magnetic separator (e.g. Invitrogen catalogue # CS 15096, or similar), and leave for 90 seconds to allow magnetic beads to form a pellet on the side of the wells. Note, if solution is viscous, longer incubations may be required.
  • a 24-pin magnetic separator e.g. Invitrogen catalogue # CS 15096, or similar
  • RNAase The following components/parameters were varied and optimised: pH, MES, NaOAc, Tris-HCl, NaCl, LiCl, GaHCl, EDTA, DTT, SMBS, ⁇ -ME, PVP, Triton X-IOO, Na Dexycholate, SDS, CTAB, sarkosyl, Proteimase K, ⁇ -amylase and RNAase.
  • Tris Systematic name 2-Amino-2-(hydroxymethyl)propane-l,3-diol
  • Trivial name trishydroxymethylamino methane
  • Buffering agent suitable for buffering more acidic solutions
  • Extraction Buffers 25mM-500mM MES pH 4.2-5.7
  • Buffering agent suitable for buffering more acidic solutions
  • Negatively charged phosphates on DNA cause molecules to repel each other.
  • the Na+ ions will form an ionic bond with the negatively charged phosphates on the DNA, neutralizing the negative charges and allowing the DNA molecules to maintain closer proximities.
  • An anionic detergent used to solubilise cellular membranes to enable access to
  • a potent reducing agent that is used to prevent oxidative degradation of DNA and oxidation of polyphenols.
  • Adsorbent of polyphenols Polyphenols in their oxidised form covalently binds to
  • PVP complexes with polyphenolic compounds through hydrogen bonding can also use polyvinyl polypyrrolidone (pvpp) - an insoluble alternative.
  • Protein hydrolysing enzyme Shows broad specificity- breaks a variety of peptide bonds and thus facilitates the isolation of high molecular weight DNA.
  • Extraction Buffers 50 ⁇ g/ml-200 ⁇ g/ml
  • a glycoside hydrolase enzyme used to break down starch into basic units
  • Extraction Buffers 400 ⁇ g per 96-well plate
  • Freeze dried Kiwifruit leaf tissue was extracted using over 200 different protocols in order to optimise the Lysis Buffer compositions. Results of the 5 variations outlined in table 1 above are shown in figures 9 to 13 below.
  • samples were amplified using PCR with a SNP marker, and separated by agarose gel electrophoresis. The gels were stained using ethidium bromide and photographed.
  • Figures 10 and 1 1 show very little improvement over figure 9 in the quality of the PCR product by increasing salt concentrations in the DNA extraction lysis buffers.
  • figures 12 and 13 both show marked improvements through the addition of the reducing agents dithiothreitol and ⁇ -mercaptoethanol, respectively.

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Abstract

L'invention concerne un procédé d'isolement d'un acide nucléique d'un matériel biologique. Le procédé comporte les étapes consistant à se procurer un matériel biologique en suspension dans une solution ; à ajuster le pH de la suspension susmentionnée pour qu'il se situe dans une plage d'environ pH 1 à environ pH 6 ; à mettre en contact l'acide nucléique de la suspension avec une matrice d'affinité capable de lier l'acide nucléique dans la plage de pH d'environ pH 1 à environ pH 6, afin de lier l'acide nucléique à la matrice d'affinité ; et à éluer l'acide nucléique à partir de la matrice d'affinité au moyen d'un tampon dont le pH est supérieur à 8, le procédé ne nécessitant pas d'étape de centrifugation ou de filtration avant l'avant-dernière étape.
PCT/NZ2007/000273 2006-09-19 2007-09-19 Procédé d'extraction d'acide nucléique WO2008035991A2 (fr)

Applications Claiming Priority (2)

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NZ54998306 2006-09-19
NZ549983 2006-09-19

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WO2008035991A3 WO2008035991A3 (fr) 2008-07-10

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010072834A1 (fr) * 2008-12-23 2010-07-01 Qiagen Gmbh Procédé de purification d'acides nucléiques
WO2014052551A1 (fr) * 2012-09-28 2014-04-03 Cepheid Procédés d'extraction d'adn et d'arn à partir d'échantillons tissulaires fixés incorporés dans de la paraffine
EP2757156A4 (fr) * 2011-09-13 2015-08-26 Sony Corp Procédé de purification d'acide nucléique, procédé d'extraction d'acide nucléique, et kit de purification d'acide nucléique
US9422543B2 (en) 2008-08-08 2016-08-23 Cambridge Enterprise Limited Isolation of nucleic acid
US10465182B2 (en) 2015-07-24 2019-11-05 Cepheid Compositions and methods for DNA and RNA extraction from tissue samples
WO2022211084A1 (fr) * 2021-04-01 2022-10-06 富士レビオ株式会社 Procédé de traitement d'un échantillon biologique

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CA2318306A1 (fr) * 1997-12-06 1999-06-17 Dna Research Instruments Limited Isolement d'acides nucleiques
GB0229287D0 (en) * 2002-12-16 2003-01-22 Dna Res Innovations Ltd Polyfunctional reagents
WO2004056984A2 (fr) * 2002-12-18 2004-07-08 University Of Geneva Procede enzymatique d'isolation d'adn de tissus vegetaux

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US9422543B2 (en) 2008-08-08 2016-08-23 Cambridge Enterprise Limited Isolation of nucleic acid
WO2010072834A1 (fr) * 2008-12-23 2010-07-01 Qiagen Gmbh Procédé de purification d'acides nucléiques
US9102935B2 (en) 2008-12-23 2015-08-11 Qiagen Gmbh Nucleic acid purification method
CN102264902B (zh) * 2008-12-23 2018-06-01 恰根有限公司 核酸纯化方法
EP2757156A4 (fr) * 2011-09-13 2015-08-26 Sony Corp Procédé de purification d'acide nucléique, procédé d'extraction d'acide nucléique, et kit de purification d'acide nucléique
US9498737B2 (en) 2011-09-13 2016-11-22 Sony Corporation Method of purifying nucleic acids, method of extracting nucleic acids and kit for purifying nucleic acids
US10023860B2 (en) 2011-09-13 2018-07-17 Sony Corporation Method of purifying nucleic acids and kit for purifying nucleic acids
WO2014052551A1 (fr) * 2012-09-28 2014-04-03 Cepheid Procédés d'extraction d'adn et d'arn à partir d'échantillons tissulaires fixés incorporés dans de la paraffine
US10233440B2 (en) 2012-09-28 2019-03-19 Cepheid Methods for DNA and RNA extraction from fixed paraffin-embedded tissue samples
US11299726B2 (en) 2012-09-28 2022-04-12 Cepheid Methods for DNA and RNA extraction from fixed paraffin-embedded tissue samples
US10465182B2 (en) 2015-07-24 2019-11-05 Cepheid Compositions and methods for DNA and RNA extraction from tissue samples
WO2022211084A1 (fr) * 2021-04-01 2022-10-06 富士レビオ株式会社 Procédé de traitement d'un échantillon biologique

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