JP2002541839A - Magnetic DNA extraction kit for plants - Google Patents

Abstract

  (57) [Summary] Methods and kits for extracting DNA from plants are provided. It rapidly produces plant DNA with a high level of purity. This method employs an anionic group, preferably immobilized on a chromatographic substrate, more preferably immobilized on magnetic beads derivatized with an anionic group (eg, dimethylaminoethyl (DEAE)). To isolate DNA (genomic, chloroplast, and / or mitochondrial DNA) via anion exchange interactions. The purified DNA is then eluted with ions (typically a salt solution). RNA can be removed by digestion with RNAse.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates generally to the field of methods and kits for the extraction of DNA,
In particular, it relates to methods and kits for extracting DNA from plants.

[0002] Methods for extracting DNA from plants include the following.

[0003] The Dellaporta method, Dellaporta, S. et al. L. Et al., 1983. Mini-preparation of plant DNA: Version II, Plant Mol Bi
ol Rep 1: 19-21. Here, the DNA is precipitated and a very coarse preparation (
Many ethanol-insoluble contaminants are present in the solution). Della
A variation of the porta method introduces an additional step of purifying the DNA via ultracentrifugation through a cesium chloride (CsCl) gradient for several hours or overnight. Rich
ards, E .; Et al., 1994, Current Protocols in M.
Molecular Biology, Addendum 27, 2.3.1-2.3.7. Cs
Purifying DNA through a Cl gradient is an old established technique where very pure DNA can be obtained, but is time consuming, expensive and produces very low yields.

[0004] An alternative DNA extraction method is cetyltrimethylammonium bromide (CTA).
B) The method. Murry, M .; G. FIG. Et al., 1980, Nucleic Acid
s Res, 8: 4321-2325. CTAB is a cationic surfactant, which forms an insoluble complex with DNA in the presence of a low concentration of salt (eg, 0.5 M sodium chloride (NaCl)) solution. In the CTAB method, the original lysis solution contains about 1.4M NaCl. DNA binds to CTAB when the NaCl concentration is reduced to 0.5M. This method is also time consuming and the procedure does not use organic solvents, so a further step to clean DNA: final extraction with organic solvents to remove preparations of polysaccharide compounds and phenolic compounds , Must be included.

[0005] Methods for the extraction of DNA from bacterial cells are more well established. Usually, cells are lysed by placing the cells in an alkaline environment. Plasmid DNA was obtained by precipitating chromosomal DNA and plasmid DN in the supernatant solution.
By leaving A in place, it is separated from the bacterial chromosomal DNA. Reev
After the chromosomal DNA has been separated from the plasmid DNA, magnetic beads are inserted into the supernatant containing the plasmid, as described in US Pat. No. 5,665,544 to E et al. The plasmid DNA binds to the beads and the plasmid DN
A is separated from contaminants in the supernatant. US Patent No. 4 to Selingson et al.
, 935, 342 discloses a general method for the isolation and purification of nucleic acids based on binding to and elution from an anion exchange column. This involves the use of successive columns containing a weak anionic exchange material and a strong anion exchange material. U.S. Patent No. 5,705,628 to Hawkins et al. Has a coating containing functional groups, specifically carboxyl groups or negatively charged groups, for purification and isolation of DNA by binding and elution. Disclosed is the use of magnetic microparticles. International application WO 96/18731 by Deggerdal et al. Describes surfactants, preferably anionic surfactants such as SDS or SARCOS.
Disclosed is a method for the isolation of nucleic acids by binding to and elution from a solid support, preferably magnetic beads, in the presence of YL ^™ )). Wooddar
U.S. Pat. No. 5,650,506 to U.S. Pat. No. 5,650,506 discloses the use of a glass filter membrane with a positively charged surface for DNA purification by binding to and elution from glass fibers.

[0006] A commercially available method for the extraction of DNA from bacterial cells is described in Annovis. Inc
. Is marketed as ISOLATE ^™ (catalog number 2-0300-85). This protocol involves the following steps: Bacterial cells are buffered (
pH 8.0), alkali-surfactant solution (0.2M N
aOH, 1% SDS) to lyse the cells, mix the suspension, neutralize the alkali and precipitate the chromosomal DNA using 3.0 M potassium acetate, pH 5.5, Centrifuging the mixture to precipitate precipitated proteins, cell debris, and denatured chromosomal DNA, plasmid DN
Mixing the supernatant containing A with the suspension containing the magnetic beads, adsorbing the DNA to the beads via anionic interaction, eluting the DNA from the beads, and reducing the salt concentration. Adjusting to 0.3, adding ethanol to make the plasmid DNA insoluble, and resulting in precipitation of the plasmid DNA from the liquid phase. However, this procedure does not work with plant genomic DNA.

[0007] It is therefore an object of the present invention to provide a method for extracting and purifying DNA from plants having a high degree of purification and integrity in a short time.

Another object of the present invention is to provide a kit having reagents for quickly and inexpensively extracting and purifying DNA from a plant.

SUMMARY OF THE INVENTION [0009] Methods and kits are provided for the extraction of DNA from plants, which rapidly purify plant DNA with high levels of purity. This method uses immobilized anionic groups via anion exchange interactions, preferably on a chromatographic substrate, or, more preferably, an anionic group such as diethylaminoethyl (DEAE) DNA (genomic DNA,
Chloroplast DNA and / or mitochondrial DNA) is isolated. The purified DNA is then eluted using ions (typically a salt solution). RNA can be removed by digestion with RNAse.

DETAILED DESCRIPTION OF THE INVENTION The following terms are used herein: anionic-having a positive charge cationic-having a negative charge nonionic-having no charge Zwitterionic-facing The same number of positive and negative charges on the terminating end, and thus the compound as a whole is neutral.

[0011] The methods described herein can be used with any plant material and have been demonstrated to be effective on the following representative types of plants: Arabidopsis buds, barley embryos, tobacco leaves , Tomato leaves, soybean hypocotyls and cultured cells, white bean hypocotyls and roots, young and old pine needles.

The process generally includes the following steps: milling plant material to produce a tissue extract, lysing plant cells, removing cell debris, and plant DNA Binding to an immobilized or insolubilized substance (eg, magnetic beads), wherein the DNA is separated into a purified form.

In a preferred method, the plant material is first ground in liquid nitrogen to a powder and then a surfactant or a non-ionic surfactant (dete)
rgent) (for example, N-lauryl sarcosine)
sine) (SARKOSYL ^™ ), TRITON ^™ or NONIDET ^™ P
-40) in the presence of a lysis buffer (0.1 M Tris-HCl, pH 8.0,
0.1 M EDTA, pH 8.0, 0.25 M NaCl and 100 μg / m
l Incubate in proteinase K). The detergent or non-ionic detergent acts to solubilize cellular components and to lyse cells. Representative surfactants are listed in Table I.

The cell debris is then removed by centrifugation and the supernatant containing DNA and other soluble cellular components is collected. The DNA is then bound to immobilized or insoluble materials (eg, magnetic beads derivatized with DEAE) having anionic groups attached thereto. This material is mixed with the supernatant bound to the DNA and then removed from the supernatant using a magnetic separator.

The beads are subsequently washed, and the DNA is then preferably purified to a final concentration of 1
. It is eluted from the beads by adding NaCl to 0M. Alternatively, DNA can be removed by binding to DEAE chromatography material or filler material. The DNA is separated by washing, centrifuging or other methods known to those skilled in the art.

The methods and kits include prior art Dellaporta and CTA.
Produces DNA with the same or better yields than the B method with the same yield as the CsCl gradient method. Particular issues of the Dellaporta method (using a CsCl column) and the CTAB method (using a cationic surfactant) are:
This is avoided by this method. The method and kit also extract and purify DNA more rapidly than the CsCl method. This method, including the precipitation step, requires a total of about 2.5 hours; without the precipitation step, it takes less than 2 hours (1 hour 50 minutes) to extract and purify the plant DNA: Approximately 1 hour for lysis, 5 minutes for binding DNA to magnetic beads, 5 minutes for washing beads, 5 minutes for separating DNA from beads, for precipitating DNA from solution Takes 30 minutes, and 40 minutes for DNA collection by centrifugation. In contrast, Cs
Cl centrifugation requires a lot of time to purify DNA.

The preferred methods described herein produce very pure DNA preparations via anion exchange interactions. Here, the DNA is replaced by chloride ions, or other negatively charged ions derived from any of a variety of salts, as binding species on the anion exchange matrix. If a traditional anion exchange column is used at the time of introducing the beads, the column appears to clog and hinders the collection of bound DNA. Alternatively, if elution is performed with a strong acid or base, significant levels of contaminants are co-eluted with the DNA. In contrast, contaminants are more easily removed than in traditional column types, resulting in a purer preparation, since the DNA bound to the beads can be mixed thoroughly with the wash solution. In addition, the DNA attached to the beads is not sheared or compressed, so that when pelleted, the attached DNA consists of longer and more intact strands.

The lysis methods for plant genomic DNA and bacterial plasmid DNA are different. The lysis method for bacterial plasmid DNA uses alkaline conditions in the presence of an anionic surfactant in the form of SDS, whereas the lysis method for plant genomic DNA uses an anionic surfactant. I do. The type of surfactant used affects the remaining steps in each method. SDS is anionic (negatively charged,
(Like DNA), it must be removed from solution in a plasmid procedure before the beads are introduced. Otherwise, SDS competes for binding to plasmid DNA on positively charged beads. In the ISOLATE ^™ plasmid extraction system, SDS is removed from solution by the addition of potassium acetate to form an insoluble precipitate with chromosomal DNA. Aggregates of SDS with bacterial genomic DNA can be easily separated from soluble plasmid DNA. In contrast, in plant systems, precipitation is not required before the binding step. The non-ionic, uncharged surfactant used in the methods and kits described herein is
This is because there is no competition for binding sites with NA. This is because genomic DNA is the desired binding species for the DEAE groups on the beads. Thus, non-ionic surfactants do not need to be removed. On the other hand, SDS and other anionic surfactants are negatively charged and compete with DNA for anionic binding sites and therefore need to be removed.

Example 1 Purification of Plant Genomic DNA The following steps describe the extraction and purification of plant-derived DNA.

1. Collect about 0.5 grams of fresh plant tissue. The tissue is deionized (d.I.
) Rinse with water to remove adhered debris and blot dry
dry).

[0021] 2. The tissue is frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle. 3
Transfer to 0 mL conical tube. Alternatively, fresh tissue may be mechanically homogenized at 4 ° C. in cold lysis buffer.

[0022] 3. 5-20 ml of lysis buffer (0.5-0.01 M per gram of starting material)
Tris-HCl, pH 7.0-8.0, 0.001-0.5M EDTA, p
H 7.0-8.0, 0.05-0.4M NaCl and 50-500 μg / m
l Proteinase K) is added to the frozen powder and mixed well.

[0023] 4. At a final concentration between 0.1% and 10%, N-lauroyl sarcosine (laur
oylsarcosine). If desired, 25-200 μg of R
NaseA can be added at this point. Alternatively, the final resuspended pellet can be treated with RNase. Incubate at 50-60 ° C. for 30 minutes to 1 hour.

5.4 Centrifuge the lysate at 5000 ° g over 5000 xg for 10 minutes to pellet debris. Transfer the supernatant to a new tube and filter, if necessary,
Remove undigested debris.

6. Add 1-30 mL of magnetic beads (40% v / v suspension) to the supernatant.
Gently mix the bead-supernatant suspension by inversion or a mechanical mixer for at least 1 minute at room temperature.

[0027] 7. Immobilize the magnetic beads on a magnetic separator and remove the supernatant.

Add between 8.5 ml and 20 ml of wash buffer, cap and remove the tube from the stand and mix gently for 5-10 minutes at room temperature.

9. Immobilize the magnetic beads on a magnetic separator and remove the supernatant. If the supernatant is substantially colored, repeat the wash.

[0029] Between 10.5 and 10 ml of elution buffer (0.7-1.5 M NaCl, 0.5
~ 0.01 M Tris-HCl, pH 7.0-8.0, 0.001-0.5 M EDTA, pH 7.0-8.0) is added to the magnetic beads and
Mix for 10 minutes.

[0030] 11. Immobilize the magnetic beads in the magnetic separator and transfer the supernatant to a new tube.

12. Add 1.0 to 2.5 times cold isopropanol. Shake well and incubate at -20 ° C for at least 10 minutes.

13. Collect the DNA by centrifugation at 10,000-20,000 × g at 4 ° C. for at least 15 minutes. Wash the pellet with 70% cold ethanol,
Then centrifuge again at 10,000-20,000 × g at 4 ° C. for at least 2 minutes. The pellet is air dried at room temperature and then dissolved in the desired buffer.

It is understood that the disclosed methods and kits are not limited to the particular methodology, protocols and reagents described as they may vary. It is also understood that technical terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Is done.

[0034]

[Table 1]

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Claims (14)

[Claims] 1. A method for extracting DNA from plant material, comprising: (a) grinding the plant material to produce a plant material extract; (b) 0.5-0.01 M Tris-HCl. , PH 7.0-8.0, 0.0
01-0.5M EDTA, pH 7.0-8.0, 0.05-0.4M NaC
lysing the plant material extract cells in a lysis buffer solution containing 1 and 50-500 μg / ml proteinase K; (c) removing the plant cell material debris; and (d) immobilizing the beads. Binding the DNA to a positively charged group that is either insoluble or soluble; (e) separating the bound DNA from the remaining cellular material; and (f) increasing the salt ion concentration of the elution buffer. Separating the bound DNA from the positively charged beads. 2. The method of claim 1, wherein said lysis buffer comprises a non-ionic detergent. 3. The method of claim 1, wherein said positively charged group is DEAE. 4. The method of claim 1, wherein said positively charged groups are on magnetic beads. 5. The method of claim 1, wherein the plant cell debris is removed by centrifugation. 6. The method according to claim 1, wherein the elution buffer is 0.7 to 1.5 M NaCl, 0.5 to 1.5 M NaCl.
0.01M Tris-HCl, pH 7.0-8.0, 0.001-0.5M
2. The method of claim 1, comprising EDTA, pH 7.0-8.0. 7. The method of claim 1, further comprising the step of adding RNAse to said lysis buffer to digest said RNA in said plant material or purified DNA. 8. A kit for use in a method for extracting DNA from plant material, comprising: (a) a plant material extract prepared by grinding the plant material; (b) 0.5 to 0.01 M Tris -HCl, pH 7.0 to 8.0, 0.0
01-0.5M EDTA, pH 7.0-8.0, 0.05-0.4M NaC
a lysis buffer solution for lysing cells in said plant material extract containing 1 and 50-500 μg / ml proteinase K, (c) a positively charged group immobilized on beads or insoluble, and (d) ) 0.7-1.5M NaCl, 0.5-0.01M Tris-HCl
PH 7.0-8.0, 0.001-0.5M EDTA, pH 7.0-8.0
An elution buffer for separating bound DNA from the positively charged group, comprising: 9. The kit according to claim 8, wherein the solution for lysing the cells is a non-ionic solution. 10. The kit according to claim 8, wherein said positively charged group is DEAE or magnetic beads immobilized on a chromatography substrate. 11. The kit according to claim 8, wherein (a) a plant material extract prepared by grinding the plant material; (b) 0.5 to 0.01 M Tris-HCl, pH 7.0 to 7.0. 8.0, 0.0
01-0.5M EDTA, pH 7.0-8.0, 0.05-0.4M NaC
lysis buffer containing 1 and 50-500 μg / ml proteinase K; (c) N-lauroyl sarcosine; (d) magnetic beads with DEAE groups on the surface; and (e) 0.7-1.5M NaCl, 0 0.5-0.01M Tris-HCl
PH 7.0-8.0, 0.001-0.5M EDTA, pH 7.0-8.0
An elution buffer comprising: a kit comprising: 12. The kit according to claim 8, further comprising RNAse in the lysis buffer. 13. The method according to claim 11, further comprising an RNAse in the lysis buffer.
The kit according to 1. 14. The method of claim 1, wherein said lysis buffer comprises N-lauroyl sarcosine.