WO2008035991A2

WO2008035991A2 - A nucleic acid extraction method

Info

Publication number: WO2008035991A2
Application number: PCT/NZ2007/000273
Authority: WO
Inventors: Michael Ronald Cook
Original assignee: Michael Ronald Cook
Priority date: 2006-09-19
Filing date: 2007-09-19
Publication date: 2008-03-27
Also published as: WO2008035991A3

Abstract

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).

Description

A NUCLEIC ACID EXTRACTION METHOD

BACKGROUND

Methods for isolating 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.

However some applications of such methods require additional pre-treatment steps to remove unwanted tissue and/or cellular debris before the nucleic acid in the sample can be contacted with the affinity matrix. For example, many procedures require precipitation and centrifugation to remove unwanted tissue debris and other non-nucleic acid materials, such as proteins and cellular membranes, before the nucleic acid can be purified from a resulting cleared lysate.

For these reasons such methods are often not amenable to automated procedures for extracting nucleic acid from multiple biological samples. Although use of plate centrifuges is possible, such apparatus is expensive. Vacuum filtration manifolds can also be used to separate fluid and solid phases. However, such apparatus can be unreliable. It would therefore be beneficial to provide an affinity-based nucleic acid extraction method that requires fewer of, or no such additional pre-treatment steps.

It is an object of the invention to provide an affinity based method for isolating a nucleic acids from biological material that does not require a precipitation or centrifugation step prior to contacting the nucleic acid from the biological material with the affinity matrix, and/or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION

In one aspect 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).

In one embodiment 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.

In a preferred embodiment the biological material is the biological material is plant tissue.

Preferably the plant tissue is freeze-dried prior to suspension in the solution of step a).

More preferably the biological material is plant leaf tissue.

Preferably the solution in a) comprises a detergent.

Preferably the solution in a) comprises a nuclease.

In one embodiment the nucleic acid is DNA.

In an alternative embodiment the nucleic acid is RNA.

In a preferred embodiment all steps are performed in the wells of multi-well plates.

In a particularly preferred embodiment all steps are performed in an automated apparatus. In a preferred embodiment the nucleic acid is eluted from the affinity matrix into a solution with a pH of more than 8, more preferably more than 9.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect 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).

1. Definitions

The term "isolating" or grammatical equivalents thereof as used herein with respect to nucleic acid means separating the nucleic acid from its normal cellular environment, to substantially purify the nucleic acid. Preferably 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.

The term "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. The term 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 term "about" as used herein means within plus or minus 10% of the specified value.

The term "comprising" or grammatical equivalents thereof as used herein means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

2. Biological material

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.

Preferably the biological material is plant tissue. More preferably the plant tissue is leaf tissue. Preferably 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.

In one embodiment 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.

3. Nucleic acid

Preferably 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. Preferably the DNA is genomic DNA.

Alternatively, the nucleic acid to be isolated is RNA. 4. The solution in step a)

pH and buffering

Preferably 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.

Preferably 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. Preferably 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. Preferably MES is included at about 15OmM.

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. Preferably 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.

When the nucleic acid to be isolated is RNA, 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. Detergent

The detergent in the solution in step a) may be anionic, non-ionic, cationic or zwitterionic or a mixture. Preferably the detergent is an anionic detergent, for example an alkyl sulphate or alkyl sulphonate.

In one embodiment the detergent in the solution in a), is SDS. Preferably 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.

In one embodiment the detergent in the solution in a), is Triton X-100. Preferably 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%. Most preferably the Triton X- 100 is at a concentration of 0.5% w/v.

In certain embodiments the solution in step a) may include two detergents. A combination of SDS and Triton X-100 is preferred.

Other suitable detergents include but are not limited to CTAB, Sodium deoxycholate and sarkosyl.

Chaotropic agent

In one embodiment the solution in step a) contains 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. Preferably 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.

Antioxidant

The solution of step a) preferably includes an antioxidant. Any suitable antioxidant may be used including for example β-mercaptoethanol, dithiothreitol (DTT) or sodium metabisulphate

(SMBS). Preferred antioxidants include β-mercaptoethanol (β-ME) and DTT.

Preferably β-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 β-ME is included at a concentration of about 0.1% w/v.

Preferably 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.

Ion-chelating agent

Preferably 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.

Preferably 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. Preferably EDTA is included at a concentration of about 25mM.

Polyphenol absorber

Preferably 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.

Preferably 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%. In addition to the components discussed above, the solution in step a) preferably contains one or more components selected from proteases, salts and solvents.

5. Dispersing the biological material in the solution of step a)

In one embodiment 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.

6. pH adjustment

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. Preferably the MES has a pH of 5.2. Preferably the MES is at a concentration of 200 mM, more preferably 10OmM, more preferably 50 mM and most preferably 25mM.

When MES is used to adjust the pH, the solution in step a) should preferably include at least one of Polyvinyl Pyrrolidone (PVP) and Sodium MetaBisulphite (SMB). Preferably both PVP and SMB should be included. Preferably both PVP and SMB should be included at 2% W/V.

7. The 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. Alternatively 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. 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.

8. Simultaneous acidification and contacting nucleic acid with the affinity matrix ^■

In one embodiment step b) and step c) are performed simultaneously. For example 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.

9. Washing

In one embodiment 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.

Preferably washing of the affinity matrix is with a solution, optionally buffered, with approximately neutral pH. Preferably 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

Preferably the buffer in step d) into which the nucleic acid is eluted has a pH in the range between pH 8 and pH 10. Preferably in the pH range 8.1 to 8.9, preferably 8.2 to 8.8, preferably 8.3 to 8.7, preferably 8.4 to 8.6 and most preferably pH 8.5. Preferably a buffer is used that will not adversely effect downstream applications of the nucleic acid, such as PCR amplification. Preferably the buffer is a Tris-HCl buffer, more preferably 10 mM Tris-HCl. Most preferably the buffer also contains EDTA, preferably at a concentration of 1 mM.

11. Use of the method in automated approaches

Preferably 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. A preferred multi-well plate format -is 96 well. Any multiwell plates could be used, preferably 96-well 1.2ml deepwell polypropelene plates are used.

Preferably the method is carried out on an automated liquid handling system. Although 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.

12. Advantages of the present invention

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. When 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.

Alternatively 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.

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.

As described above, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with reference to the accompanying drawings in which:

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).

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.

A) LBl : 125μl Invitrogen Chargeswitch L18 buffer

25μl 10% SDS 2μl Invitrogen Chargeswitch RNase

LB2: 125μl Invitrogen Chargeswitch Ll 8 buffer

B) As in A above, but LB2 also contains 1 μl cone HCl

C) As in A above, but LB2 also contains 2μl cone HCl

D) As in A above, but LB2 also contains 2.5 μl cone HCl E) As in A above, but LB2 also contains 3 μl cone HCl

F) As in A above, but LB2 also contains 5μl cone HCl

20μl loaded per sample. 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.

A) LBl : 125μl Invitrogen Chargeswitch Ll 8 buffer 25μl lO% SDS

2μl Invitrogen Chargeswitch RNase LB2: 125μl Invitrogen Chargeswitch Ll 8 buffer 5μl cone HCl

B) As in A above, but LBl and LB2 also contain 1% Polyvinyl pyrrolidone (PVP) C) As in A above, but LBl and LB2 also contain 2% PVP

D) As in A above, but LBl and LB2 also contain 1% Sodium Metabisulphite (SM)

E) As in A above, but LBl and LB2 also contain 1% PVP and 1% SM

20μl loaded per sample.

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

2.

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).

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 β-mercaptoethanol (BME). EXAMPLES

The invention will now be illustrated with reference to the following non-limiting examples.

Example 1: Isolation of nucleic acid by a "manual" protocol of the method of the invention using pH-based reversible binding of nucleic acid

Biological material

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. - 95⁰C 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 -20⁰C with desiccated silica until required.

Methods

1. Prepare enough Lysis Buffer 1 (LBl) for all samples by combining the following volumes for each sample: 250μl of Invitrogen buffer L 18, 2μl of Invitrogen RNase (or similar) and 25μl of a 10% SDS solution.

2. Prepare enough Magnetic Beads (MB) for all samples by combining the following volumes for each sample: 40μl Invitrogen Magnetic Beads, 20μl of Invitrogen Dl, 5μl concentrated HCl.

3. Pipette 277μl of LBl into the appropriate well of a 1.2ml x 96 deep- well plate containing 10-50mg (wet weight) of freeze dried plant tissue (e.g. leaf) that has been finely ground using a suitable grinding mill (e.g. Tissue Disintegrator by A-Tech (NZ) - 2 x lmin at 32Hz). Aspirate and dispense several times to resuspend ground leaf material, being careful not to introduce bubbles.

4. Incubate at room temperature for 5 minutes

5. Aspirate 200μl of the homogenate and transfer to a clean microtitre plate.

6. Pipette 65μl of MB into the tissue homogenate, and mix briefly, avoiding bubble formation 7. Incubate for 1 minute at room temperature

8. 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.

10. Remove the microtitre plate from the magnetic separator, and resuspend the pellet thoroughly with 200μl of distilled water, or Invitrogen Wash Buffer.

1 1. Repeat steps 8-10 once more

12. Repeat step 8 and 9 13. Resuspend pelleted beads with 150μl of 1OmM Tris-HCl (pH 8.5), ImM EDTA (pH 8.0), or Invitrogen elution buffer

14. Incubate for 1 minute at room temperature.

15. Place the microtitre plate on a magnetic separator for 90 seconds, or until magnetic beads have formed a firm pellet. 16. Aspirate all liquid (containing nucleic acid) and transfer to final storage plate.

All solutions labelled "Invitrogen" refer to those supplied in the ChargeSwitch® gDNA Plant kit, catalog number CS 18000- 10, provided by Invitrogen Life Technologies, USA

The nucleic acid extraction procedures described above were followed, and compared with the original ChargeSwitch Invitrogen protocol.

Results

20μl of the nucleic acid solution from eight duplicate tissues samples produced were electrophoresed on a 0.9% agarose slab gel at 70 volts for approximately 30 minutes. 50ng lambda standards were included for an estimation of nucleic acid yield. The gel was stained in ethidium bromide. Results are shown in Figure 1. DNA samples from the manual protocol of the invention (above) as shown in row 2, and demonstrate a compatible yield to that obtained using the ChargeSwitch samples in row 1.

Example 2: Isolation of nucleic acid by an automated protocol

Biological material Apple leaf material was prepared as described in Example

Method

1. Prepare Lysis Buffer 1 (LBl) for all samples by combining the following volumes for each sample: 125μl Invitrogen buffer L 18, 25μl 10% SDS and 2μl Invitrogen RNase (or similar).

2. Prepare Lysis Buffer 2 (LB2) for all samples by combining the following volumes for each sample: 125μl Invitrogen buffer L 18, 5μl concentrated HCl

3. Prepare Magnetic Beads (MB) for all samples by combining the following volumes for each sample: 40μl Invitrogen Magnetic Beads, 20μl Invitrogen Dl.

4. Add 152μl of LBl to the appropriate well of a 1.2ml x 96 deep-well plate containing 10- 50mg (wet weight) of freeze dried plant tissue (e.g. leaf) that has been finely ground using an appropriate grinding mill (e.g. Tissue Disintegrator by A-Tech (NZ) - 2 x lmin at 32 Hz). Aspirate and dispense several times to resuspend ground leaf material, being careful not to introduce bubbles.

5. Transfer the deep- well plate to a plate shaker and shake for 1 minute to assist resuspension of plant tissue (e.g. Variomag Teleshake plate shaker). 6. Incubate at room temperature for 5 minutes

7. Add 130μl of LB2 and mix by aspirating and dispensing (being careful not to introduce bubbles) before transferring 200μl of the homogenate to a fresh microtitre plate.

8. Add 60μl of MB and transfer the plate to the plate shaker for 1 minute to assist dispersion of magnetic beads throughout the tissue homogenate. 9. Incubate at room temperature for 1 minute.

10. 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.

1 1. Aspirate all liquid from the wells, ensuring the pellet remains intact. 12. Remove the microtitre plate from the magnetic separator, and resuspend the pellet thoroughly with 200μl of distilled water, or Invitrogen Wash Buffer.

13. Repeat steps 10-12 once more.

14. Repeat step 10 and 11. 15. Resuspend pelleted beads with 150μl of 1OmM Tris-HCl (pH 8.5), ImM EDTA (pH 8.0), or Invitrogen elution buffer

16. Incubate for 1 minute at room temperature.

17. Place the microtitre plate on a magnetic separator for 90 seconds, or until magnetic beads have formed a firm pellet.

18. Aspirate all liquid (containing nucleic acid) and transfer to final storage plate.

Results

20μl of the nucleic acid solution from eight duplicate tissues samples produced were electrophoresed on a 0.9% agarose slab gel at 70 volts for approximately 30 minutes. 50ng lambda standards were included for an estimation of nucleic acid yield. The gel was stained in ethidium bromide. Results are shown in Figure 1.

DNA samples from the "automated" protocol of the invention (above) as shown in row 3 and demonstrate a compatible yield to that obtained using the ChangeS witch protocol (row 1) and the "manual" protocol of the invention (Example 2) shown in row 2.

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.

The applicants used the protocol below for this example.

Protocol for robot

1. Add 198μl of LBl (Lysis Buffer 1) to the appropriate well of a 1.2ml x 96 deep-well plate containing 10-50mg (wet weight) of freeze dried plant tissue (e.g. leaf) that has been finely ground using an appropriate grinding mill (e.g. Tissue Disintegrator by A-Tech (NZ) - 2 x lmin at 32 Hz). 2. Transfer the deep-well plate to a plate shaker and shake for 1 minute to assist resuspension of plant tissue (e.g. Variomag Teleshake plate shaker).

3. Add 198μl of LB2 (Lysis Buffer 2) to each well .Aspirate and dispense several times to resuspend ground leaf material, being careful not to introduce bubbles, before transferring 150μl of the homogenate to a fresh microtitre plate. 4. Add 50μl of MBM (Magnetic Bead mix) and transfer the plate to the plate shaker for 1 minute to assist dispersion of magnetic beads throughout the tissue homogenate.

5. Incubate at room temperature for 1 minute.

6. 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.

7. Aspirate all liquid from the wells, ensuring the pellet remains intact.

8. Remove the microtitre plate from the magnetic separator, and resuspend the pellet thoroughly with 200μl of distilled water, or Invitrogen Wash Buffer. 9. Repeat steps 6-8 once more.

10. Repeat step 6 and 7.

1 1. Resuspend pelleted beads with 150μl of 1OmM Tris-HCl (pH 8.5), ImM EDTA (pH 8.0), or Invitrogen elution buffer

12. Incubate for 1 minute at room temperature. 13. Place the microtitre plate on a magnetic separator for 90 seconds, or until magnetic beads have formed a firm pellet. 14. Aspirate all liquid (containing DNA) and transfer to final storage plate

NOTE: The washing to elution steps (6 on to 1 1) are done on a microplate washer complete with 2 sets of 96-well pins for dispensing and aspirating.

The applicants trialled modifications to the Invitrogen ChargeSwitch buffers in an attempt to establish a working protocol for the method of the invention on a robot.

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.

2% PVP or/and 2% SM was optionally added just before use

Results The resulting DNA was electrophoresed as described in Example 2.

Results are shown in Figures 7 and 8 for HCl addition and PVP and SM addition respectively.

The best results were achieved with 5ul of HCl and 1 or 2% of either PVP or SM or a combination of both.

Example 4: Validation of the usefulness of the nucleic acid isolated by the method of the invention in downstream applications

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..

The products of the automated protocol shown above were diluted 32-fold, and used directly as template in PCR reactions. Two controls were included, consisting of template nucleic acid that was extracted using a conventional manual chloroform extraction protocol. The resulting PCR products were electrophoresed at 1 10 volts for approximately 90 minutes on 0.9% agarose gels, and stained using ethidium bromide:

The results in Figure 2 clearly show that the nucleic acid isolated by the method of the invention is suitable for downstream PCR applications. 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.

Example 5: Use of alternative buffers in the methods of the invention

Apple leaf material was prepared as described in Example 1.

As an alternative to use of LB2 in the "automated" protocol of the invention described in Example 2 MES pH 5.2 was trialled at a range of concentrations ((25mM, 5OmM, 10OmM and 20OmM). In the same experiment 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 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.

The "automated" protocol of Example 2 was performed using the modifications described above.

The resulting isolated DNA was electrophosed, as described in Example 2, and the results without PVP/SMB are shown in Figure 3.

Results with PVP/SMB are shown in Figure 4.

The addition of PVP and SMB provided significantly better yields than those trials omitting them. Estimated yield is around l-1.5ng/μl ,in a final volume of 150ul, which is ideal for direct use as template in PCR reactions.

All samples were also used directly as template (2μl) in 17μl PCR reactions using a RAPD primer, and electrophoresed at HOv for approx 90 minutes on 0.9% agarose slab gels that were subsequently stained in ethidium bromide and visualised under UV:

Results without PVP/SMB are shown in Figure 5. Results with PVP/SMB are shown in Figure 6.

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.

Example 6: Optimisation of solutions for the method of the invention

Apple leaf material was prepared as described in Example 1. The applicants optimised the components and pH of the solution from step a) of the method of the invention using the protocol of the method shown below.

Protocol for robot

I . Add 198μl of LBl (Lysis Buffer 1) to the appropriate well of a 1.2ml x 96 deep- well plate containing 10-50mg (wet weight) of freeze dried plant tissue (e.g. leaf) that has been finely ground using an appropriate grinding mill (e.g. Tissue Disintegrator by A-Tech (NZ) - 2 x lmin at 32 Hz). 2. Transfer the deep- well plate to a plate shaker and shake for 1 minute to assist resuspension of plant tissue (e.g. Variomag Teleshake plate shaker). 3. Add 198μl of LB2 (Lysis Buffer 2) to each well .Aspirate and dispense several times to resuspend ground leaf material, being .careful not to introduce bubbles, before transferring

150μl of the homogenate to a fresh microtitre plate. 4. Add 50μl of MBM (Magnetic Bead mix) and transfer the plate to the plate shaker for 1 minute to assist dispersion of magnetic beads throughout the tissue homogenate.

5. Incubate at room temperature for 1 minute.

7. Aspirate all liquid from the wells, ensuring the pellet remains intact.

8. Remove the microtitre plate from the magnetic separator, and resuspend the pellet thoroughly with 200μl of distilled water, or Invitrogen Wash Buffer.

9. Repeat steps 6-8 once more. 10. Repeat step 6 and 7.

I I . Resuspend pelleted beads with 150μl of 1OmM Tris-HCl (pH 8.5), ImM EDTA (pH 8.0), or Invitrogen elution buffer

12. Incubate for 1 minute at room temperature.

13. Place the microtitre plate on a magnetic separator for 90 seconds, or until magnetic beads have formed a firm pellet.

14. Aspirate all liquid (containing DNA) and transfer to final storage plate.

NOTE: The washing to elution steps (6 on to 1 1) are done on a microplate washer complete with 2 sets of 96-well pins for dispensing and aspirating. Approximately 200 different solutions were trialled which correspond to the solution of step a) (extraction buffer), a wash buffer and elation buffer.

The following components/parameters were varied and optimised: pH, MES, NaOAc, Tris-HCl, NaCl, LiCl, GaHCl, EDTA, DTT, SMBS, β-ME, PVP, Triton X-IOO, Na Dexycholate, SDS, CTAB, sarkosyl, Proteimase K, α-amylase and RNAase.

A summary of the results including the useful range of each component in the method of the invention is provided below:

1.0 Buffer

1.1 Tris Systematic name: 2-Amino-2-(hydroxymethyl)propane-l,3-diol Trivial name: trishydroxymethylamino methane

Use: Used as a component for buffering solutions. It has an effective pH range between 6.5 and 9.7, thus making it an effective buffer for slightly basic solutions to keep DNA deprotonated and soluble in water. Note, this can be used for LBl, as long as LB2 brings the pH down sufficiently for DNA to bind. Range: Extraction Buffers— 25mM-250mM Tris pH 8.0 Wash Buffers — N.A

Elution Buffers — 5mM-l 5OmM Tris pH 8.5-9

1.2 MES

Systematic name: 2-(/V-morpholino)ethanesuIfonic acid Trivial name: N.A.

Use: Buffering agent suitable for buffering more acidic solutions

Range: Extraction Buffers— 25mM-500mM MES pH 4.2-5.7

1.3 NaOAc

Systematic name: Sodium Acetate Trivial name:

Use: Buffering agent suitable for buffering more acidic solutions

Range: Extraction Buffers — 15OmM-IM with pH 4.0-5.7

2.0 Salts

2.1 NaCl

Systematic name: Sodium Chloride Trivial name: salt

Use: Helps in removal of polysaccharides by increasing their solubility. 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.

Range: Extraction Buffers — 250mM-2.5M

2.2 GuHCl

Systematic name: Aminomethanamidine hydrochloride Trivial name: Guanidine hydrochloride

Use: Strong chaotropic agent useful for the denaturation and subsequent refolding of proteins. Urea and DTT may also be needed. Range: Extraction Buffers — 0.7M-7M

3.0 Detergents

3.1 Triton X 100

Systematic name: polyethylene glycol p-(l,l,3,3-tetramethylbutyl)-phenyl ether Trivial name: Triton X 100

Use: A non-ionic detergent used for solubilising cellular membranes to enable access to nucleic acids. Range: Extraction Buffers— 0.25%-4%

3.2 Na Deoxycholate

Systematic name: Sodium Deoxycholate Trivial name:

Use: Biological detergent used for lyse of cells and to solubilise cellular membrane components

Range: Extraction Buffers— 0.5%-4%

3.3 SDS

Systematic name: Sodium dodecyl sulfate

Use: an ionic detergent also used for destroying structural organisation of protein Range: Extraction Buffers— 0.5%-4%

3.4 CTAB

Systematic name: Cetyl trimethylammonium bromide

Use: a cationic surfactant/detergent. At high salt concentration forms insoluble complexes with proteins and most acidic polysaccharides leaving nucleic acids in solution Range: Extraction Buffers— 0.5%-4%

3.5 Sarkosyl Systematic name: Sodium Lauryl Sarcosinate Trivial name: Sarkosyl

Use: An anionic detergent used to solubilise cellular membranes to enable access to

Nucleic acids.

Range: Extraction Buffers— 0.5%-4%

4.0 Additives 4.1 EDTA

Systematic name: ethylenediamine tetraacetic acid Trivial name:

Use: an ion-chelating agent commonly used to inactivate metal-dependent enzymes which could damage DNA (e.g. nucleases). Range: Extraction Buffers — 5mM-50mM EDTA

4.2 DTT Systematic name:

Trivial name: dithiothreitol

Use: An antioxidant that helps to minimise oxidative degradation of nucliec acids during the extraction process. Polyphenols only bind to DNA when in their oxidized state. DTT is an antioxidant and thus prevents the oxidation of polyphenols.

Range: Extraction Buffers — 10mM-50mM

4.3 SMBS Systematic name: Sodium metabisulphite

Use: A potent reducing agent that is used to prevent oxidative degradation of DNA and oxidation of polyphenols.

Range: Extraction Buffers— l%-4%

4.4 β-Me

Systematic name: β-mercaptoethanol

Trivial name:

Use: Polyphenols only bind to DNA when in their oxidized state. β-Me is an antioxidant and thus prevents the oxidation of polyphenols. β-Me also prevents degradative oxidation of nuclei acids, polymerisation of tannins and also helps destroy structural organisation of proteins which may be present in the lysate. Range: Extraction Buffers— 0.1%-0.5%

4.5 PVP Systematic name: Poly vinyl pyrrolidone Trivial name: PVP

Use: Adsorbent of polyphenols (Polyphenols in their oxidised form covalently binds to

DNA and renders it useless for downstream processes, e.g. PCR). PVP complexes with polyphenolic compounds through hydrogen bonding (separates polyphenols from DNA). Note, can also use polyvinyl polypyrrolidone (pvpp) - an insoluble alternative.

Range: Extraction Buffers— l%-4%

5.0 Enzymes

5.1 Proteinase K

Systematic name: Trivial name:

Use: Protein hydrolysing enzyme. Shows broad specificity- breaks a variety of peptide bonds and thus facilitates the isolation of high molecular weight DNA.

Range: Extraction Buffers — 50μg/ml-200μg/ml

5.2 α-amylase Systematic name: Trivial name:

Use: A glycoside hydrolase enzyme used to break down starch into basic units

(glucose). Carbohydrates are inhibitory for downstream applications such as PCR and commonly co-purify with DNA. Range: Extraction Buffers - 10units-500units

5.3 Rnase

Systematic name: Trivial name:

Use: degrade RNA within the lysate

Range: Extraction Buffers — 400 μg per 96-well plate

5.4 pH :

Lysis buffer can be any pH, as long as the final solution pH is appropriate for binding, e.g. 4-6.5 ideal = 5.2. i.e. LBl could be basic, and LB2 could be acididc to bring overall pH down to approximately 5 for DNA binding.

washing 4-7 ideal = 6.8

elution 7.5-10 ideal 8.5-9.0

Particularly beneficial combinations of components (kits) for the solutions of the buffer in step a) are shown in table 1 below.

Table 1

SDS 0.5% 0.5% 0.5% 0.5% 0.5%

Results

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.

Following the DNA extraction, samples were amplified using PCR with a SNP marker, and separated by agarose gel electrophoresis. The gels were stained using ethidium bromide and photographed.

Results are shown. in Figures 9 (Kit 43), 10 (Kit 182), 11 (Kit 183), 12 (Kit 69) and 13 (Kit 44).

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. However, figures 12 and 13 both show marked improvements through the addition of the reducing agents dithiothreitol and β-mercaptoethanol, respectively.

The above examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.

Claims

CLAIMS:

1. 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).

2. The method of claim 1 in which 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.

3. The method of claim 1 or 2 in which the biological material is plant tissue.

4. The method of any one of claims 1 to 3 in which the biological material is plant leaf tissue.

5. The method of any one of claims 1 to 4 in which the biological material is plant leaf tissue is freeze-dried prior to suspension in the solution of step a).

6. The method of any one of claims 1 to 5 in which the nucleic acid is DNA.

7. The method of any one of claims 1 to 6 in which the nucleic acid is RNA.

8. The method of any one of claims 1 to 7 in which all steps are performed in multi-well plates.

9. The method of any one of claims 1 to 8 in which all steps are performed in an automated apparatus.

10. The method of any one of claims 1 to 9 in which the solution in a) comprises a detergent.

1 1. The method of any one of claims 1 to 10 in which the solution in a) is buffered.

12. The method of any one of claims 1 to 1 1 in which the solution in a) comprises an antioxidant.

13. The method of any one of claims 1 to 12 in which the solution in a) comprises a ion- chelating agent.

14. The method of any one of claims 1 to 13 in which the solution in a) comprises a polyphenol absorber.