AU645630B2 - DNA isolation method - Google Patents

Description

64 130 S F Ref: 195961

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Pioneer Hi-Bred International, Inc.

700 Capital Square 400 Locust Square Des Moines Iowa 50309 UNITED STATES OF AMERICA Anil K. Jhingan Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia DNA Isolation Method The following statement is a full description of this invention, including the best method of performing it known to me/us:i.

i Technical Field This invention relates to the isolation of DNA from whole plants and plant cells, tissues and parts, and from yeasts and bacteria.

c~ Background of the Invention With the increasing need for DNA fingerprinting, restriction fragment length polymorphism (RFLP) analysis, Southern transfers, construction of genomic libraries and t.raisformation experiments in biotechnology, the isolation high molecular weight (HMW) DNA becomes a major problem.

Several procedures for the isolation of HMW DNA have been reported, all of which have drawbacks for various reasons.

The methods generally involve physical grinding of cells or tissue followed by extraction in buffers containing EDTA, Tris and other reagents. Some of the reagents used react with various cellular organelles; the function of others is unknown. The prior art methods are often time consuming, irreproducible and give variable yields of DNA, involving more art than science. The DNA ,oobtained also varies in terms of its purity, and all of the methods involve purification of DNA with phenol, a protein denaturant which can be hazardous to users. Finally, a method that is effective in. DNA extraction in one plant S• group often fails when used on other plants.

More recently, a solid phase extraction material comprising silica and having hydroxyl groups on its surface has been reported as a replacement for phenol for removal of proteins. However, the preparation of this material is cumbersome, and grinding of tissue is still needed.

c In view of these difficulties, a continuing need exists for a versatile method that would overcome these problems.

I-

2 It is an object of this invention to provide such a method.

Disclosure of the Invention According to a first embodiment of this invention there is provided a method for disrupting cell walls in a DNA extraction process, comprising the steps of contacting cells having DNA with an aqueous solution of a xanthate-forming compound of the formula /s (RO)nC

SM

wherein n is 0 or 1; R is unsubstituted or substituted lower alkyl, lower alkenyl, or aralkyl; and M is alkali metal or ammonium where n is 1 and a sulfur-carbon bond when n is 0; and isolating DNA from the cells.

While not intending to be limited by theory, the isolation of DNA from plant!, yeasts and bacteria is difficult partly due to the presence of a rigid cell wall which is rich in polysaccharides and therefore difficult to rupture completely with commonly used buffers.

Removal of the cell wall by enzymes is tedious and not always feasible. Variations in DNA yield and quality from extraction to extraction using current methods probably arises from the varying degrees of cell wall break up. Thus, there has been a need for new technique for disrupting cell walls by a thorough, yet delimited mechanism to allow isolation of DNA in a reproducible manner without the need to homogenize cells or tissues.

Polyhydric alcohols, including cellulose, have been solubilized in the past by conversion to metal xanthates. This method was discovered by Zeise in 1815 and it has been widely employed in the textile industry. Xanthates find extensive application in the separation and quantitative determination of numerous metal ions by taking advantage of the low and differential solubilities of metal xanthates under controlled pH conditions.

It has now been determined that the replacement of existing reagents for DNA extraction by xanthate-forming compounds is feasible and highly advantageous. It was postulated that these compounds would dissolve the cell wall in plants by forming water soluble polysaccharide xanthates with the hydroxyl groups of polysaccharides which make up a S substantial portion of plant cell walls. The reaction of xanthate-forming compounds with amines is also reported. Furthermore, xanthate forming compounds can also bind metal ions to inhibit DNAase activity. As a result, these compounds enable selectively dissolving DNA 30 from cell organelles, leaving contaminating proteins, metal ions and i <0o IN:VBTT)0006:LMM other compounds as an insoluble residue; DNA can then be precipitated from the supernatant.

Xanthate-forming compounds The "xanthate-forming compounds" of this invention S include any compound capable of forming xanthate reaction products with cell wall polysaccharides from plant cells.

These specifically include carbon disulfide and its organoalkaline derivatives. While the common reagent used in industrial use of this reaction (the viscose rayon ioprocess) is carbon disulfide, for analytical isolation of DN,A according to this invention 'the organoalkaline derivatives of carbon disulfide are preferred. By "organoalkaline derivatives of carbon disulfide" is meant compounds of the general formula

S

iS RO-C

SM

4, wherein R is an unsubstituted or substituted alkyl, alkenyl or aralkyl group, preferably selected from methyl, ethyl, "I propyl, butyl, hexyl, isoamyl, vinyl, allyl, 2-3d.ihydroxypropyl, phenethyl, 4-morpholinylmethyl, and S 0 hydroxyphenethyl; and wherein M is an alkali metal or NH 4 preferably Na or K. These compounds are formed by reaction of carbon disulfide with the corresponding alcoholic alkali:

CS

2 MOH ROH ROC(S)SM +H 2 0.

The most preferred of these compounds, the carbonodithioic acid o-ethyl ester, sodium salt (R=C 2

H,

M=Na; sodium ethyl xanthogenate) can be prepared by standard 1 methods, and its potassium analogue is commercially S available from Fluka. The entire class of commpounds useful in this invention (including carbon disulfide) can thus be S S 3 represented by the formula

S

(RO) C

SM

wherein n is 0 or 1; R is an unsubstituted or substituted alkyl, alkenyl or aralkyl group, preferably selected from methyl, ethyl, propyl, butyl, hexyl, isoamyl, vinyl, allyl, 2-3-dihydroxypropyl, phenethyl, 4-morpholinylmethyl, and hydroxyphenethyl; and wherein M is alkali metal or ammonium, preferably Na or K, when n is 1 and another bond to the carbon when n is 0.

e1 The methods described herein using these compounds enable efficient DNA isolation without homogenizing tissues and without removing proteins.

Example I Tissue Grinding Protocol Fresh leaf material (0.6 0.63 g) of thirteen day old corn seedlings was frozen in a liquid nitrogen bath until it was very brittle and was ground to a fine powder using a glass homogenizer. The powder was suspended in 4 ml buffered extraction reagent (0.0694 mM carbonodithioic acid, ,P o-ethyl ester, sodium salt, 100 mM Tris, pH 7.5, 700 mM NaCI, 10 mM EDTA, pH 8 or 0.0625 mM carbonodithioic acid, oethyl ester, potassium salt, 100 mM Tris, pH 7.5, 700 mM NaCl, lU mM EDTA) in 15 ml propylene tube. After 5 min.

incubation at 650, the leaf debris was removed by filtering jS the homogenate through Miracloth. The DNA was precipitated from the filtrate by addition of two volumes of ethanol and S centrifuged for 10 minutes at 3K at 40. The pellet was suspended in 100 pl TE and centrifuged for 3 min. as before to remove precipitated proteins and metal xanthates. The S3o supernatant was transferred into 1.5 ml Eppendorf tube and centrifuged for 5 minutes. The DNA was precipitated again from the supernatant by adjusting to 2M NH 4 OAC and adding 4 two volumes of ethanol. DNA was pelleted by centrifuging for 5 min. at 735 g. After decanting the supernatant, the pellet was dried in a speed vac and redissolved in 100 pl TE buffer. The yield of DNA was 20-40 pg.

SExample II Non-Grinding Protocol 1 g of fresh leaves in 4 ml of extraction buffer containing carbonodithioic acid, o-ethyl ester, sodium salt are incubated at 650 for 20 min. and filtered. The DNA is io precipitated from the filtrate and reprecipitated as above.

This non-grinding method applied to corn yielded 2.56 to 6.68 pg DNA per gram of leaf tissue.

Example III To evaluate the protocols of Examples I and II, DNA It isolated was digested for 6 h with Bam HI and Hind III, EcoRI and Sst I and assayed by agarose gel electrophoresis.

The undigested DNA showed an apparent molecular weight greater than the X marker which is 23 kb. The absence of high molecular weight DNA and presence of smear in the ao-digested samples suggested that DNA was completely digested and was free of contaminants which interfere with restriction enzyme digestion.

Example IV The. quality of the DNA preparations wls further Q, assessed by Southern transfer experiments. Isolated DNA was digested with Bam, HI, electrophoresed, transferred to MSI membrane and hybridized with 32 p single copy probes.

Undigested and digested DNA gave the expected hybridization pattern. The appearance of discrete bands in .o the digested samples confirmed that the DNA was digested completely by the enzyme and that the hybridization with the probe was successful. This is an important criterion for the quality of DNA.

5 Example V To further substantiate the quality of the isolated DNA for molecular biology applications, extracted DNA was assayed by polymerase chain reaction (PCR). After isolation, the DNA was amplified and the products were run on an agarose gel. A control experiment was also performed in which template DNA was not included in the PCR reaction.

The absence of the expected target band in the control and its presence in the DNA samples obtained from the foregoing io protocols further confirmed the quality of DNA.

Example VI The yield and efficiency of these extraction procedures was tested with a grinding protocol. Addition of a known amount (20 pg) of DNA to the leaf sample prior to homogenization and following the same steps yielded at least S 81% DNA in the final step. This suggested that losses of DNA due to enzymatic or mechanical degradation were minimum.

Examples VII XII The grinding method has also been successfully employed at for the isolation of DNA from thirteen day old seedlings of soybean, sorghum, sunflower, alfalfa and tobacco as determined by agarose gel electrophoresis and Southern transfers. Results are shown in Table 1.

6 Table 1 Ex. Plant Yield 1 High DNA Quality 2 Southern Blot VI Alfalfa 15-42 Yes works VIII Canola 8-14 Yes IX Sorghum 12-28 Yes works X Soybean 26-37 Yes works XI Sunflower 7-30 Yes XII Tobacco 7-30 Yes i.g/600-630 mg fresh leaves lo 2 DNA is completely digested by Bam HI Examples XIII XXIV The versatility of these two methods (grinding and nongrinding) was also compared on alfalfa, barley, canola, sorghum, soybean, sunflower, tobacco, wheat, petunia, 1 ?Tspinach, yeast and E. coli. With yeast and E. coli, homogenization was omitted in the grinding protocol. Table

I

I S 7 2 gives the yields of DNA.

Table 2 Yield 1 Yield 2 Ex. Plant grinding method non-grinding method IT XIII Alfalfa 15-42 1.50-2.80 XIV Canola 8-14 2.70-4.80 XV Sorghum 12-28 1.70-2.66 XVI Soybean 26-37 0.45-1.14 XVII Sunflower 7-30 0.13-1.34 IO XVIII Tobacco 7-30 1.00-3.74 XIX Petunia 11-19 2.07-2.27 XX Lettuce 18-43 1.63-2.17 3 XXI Wheat 7-38 1.12-4.27 XXII E. coli 50 22-25 S Different Series: XXIII Spinach 20.64 1.4346 XXIV Yeast 1.239 2.369 I/g/600-630 mg fresh tissue 2 pg DNA/1 g fresh tissue Cc3pg DNA/2 g (market-purchased) lettuce Examples XXV-XIX The method of this invention was also applied successfully for the isolation of DNA from the following plants: Ex. Plant A5 XXV Celosia XXVI Alyssum The simplicity of the non-grinding method may facilitate automation of DNA isolation and field use of analytical and diagnostic methods requiring DNA isolation by Aonon-specialists. The wide applicability of the grinding method of this invention makes it a potential general method 8 of DNA isolation from plant cells. The extractions have been attempted at various temperatures using different concentrations of substrates under various pH values, using different amounts and concentrations of buffer. With the non-grinding method, alfalfa, corn, sorghum and lettuce gave high yield and quality of DNA using 2 ml of buffer/reagent.

On the other hand, isolation of DNA from soybean, sunflower and wheat using sodium ethyl xanthogenate required twice that amount to give clean DNA. With canola, tobacco and IC petunia, slight gentle homogenization prior to incubation helped to give better quality and yield of DNA. Thus, it can be seen that numerous specific embodiments of the methods of this invention can be optimized to suit the specific in vivo or in vitro system under consideration.

i i C S 9

Claims (10)

1. A method for disrupting cell walls in a DNA extracti n process, comprising the steps of contacting cells having DNA with an aqueous solution of a xanthate-forming compound of the formula -s (RO)nC SM wherein n is 0 or 1; R is unsubstituted or substituted lower alkyl, lower alkenyl, or aralkyl; and M is alkali metal or ammonium where n is 1 and a sulfur-carbon bond when n is 0; and isolating DNA from the cells.

2. The method of claim 1, wherein the xanthate-forming compound forms xanthate reaction products with cell wall polysaccharides.

3. A method according to claim 1 or 2, wherein n is 1, R is unsubstituted or substituted lower alkyl, and M is Na or K.

4. A method according to any one of claims 1 to 3, wherein R is selected from the group consisting of methyl, ethyl, propyl, butyl, hexyi, isoamyl, and 2-3-dihydroxypropyl.

5. A method according to claim 4, wherein the xanthate-forming compound is the sodium or potassium salt of carbonodithioic acid o-ethyl ester.

6. A method according to any one of claims 1 to 5, further comprising the step of filtering cellular debris from the solution.

7. A method according to claim 1, further coraprising the step of precipitating the DNA from the filtered solution with ethanol.

8. A method as defined in any one of claims 1 to 7, wherein the cells are selected from the group consisting of plant cells, yeast cells and bacterial cells.

9. A method for disrupting cell walls in a DNA extraction process, substantially as Shereinbefore described with reference to any one of the Examples. 25

10. DNA isolated from plant yeast or bacterial cells by the method of any one of claims 1 to 8. Dated 29 October, 1993 Pioneer Hi-Bred International, Inc. Patent Attorneys for the Applicant/Nominated Person 30 SPRUSON FERGUSON t l (N:JBTTI00066:LMM DNA ISOLATION METHOD Abstract A novel method for the isolation of high molecular weight DNA from plants, yeast and bacteria using xanthate- forming compounds such as sodium/potassium ethyl xanthogenate is disclosed. The procedure does not require deproteination and yields clean DNA that is suitable for both PCR and Southern blotting. It can be utilized on a small scale without homogenizing the tissue. These features Io.also facilitate automated screening of plant tissue samples, one of the labor-intensive techniques in molecular biology. This method is also adaptable for use in the field. .jh0 4.sS.O *O 0* o