CA2370656A1 - Magnetic dna extraction kit for plants - Google Patents
Magnetic dna extraction kit for plants Download PDFInfo
- Publication number
- CA2370656A1 CA2370656A1 CA002370656A CA2370656A CA2370656A1 CA 2370656 A1 CA2370656 A1 CA 2370656A1 CA 002370656 A CA002370656 A CA 002370656A CA 2370656 A CA2370656 A CA 2370656A CA 2370656 A1 CA2370656 A1 CA 2370656A1
- Authority
- CA
- Canada
- Prior art keywords
- dna
- plant material
- kit
- lysis buffer
- positively charged
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
A method and kit for the extraction of DNA from plants is provided, which quickly yields plant DNA with a high level of purity. The method isolates DNA
(genomic, chloroplast, and/or mitochondrial DNA) using immobilized anionic groups, preferably on a chromatographic substrate or more preferably magnetic beads derivatized with anionic groups such as diethylaminoethyl (DEAE) via an anion-exchange interaction. The purified DNA is then eluted with ions (typically a salt solution). RNA can be removed by digestion with RNAse.
(genomic, chloroplast, and/or mitochondrial DNA) using immobilized anionic groups, preferably on a chromatographic substrate or more preferably magnetic beads derivatized with anionic groups such as diethylaminoethyl (DEAE) via an anion-exchange interaction. The purified DNA is then eluted with ions (typically a salt solution). RNA can be removed by digestion with RNAse.
Description
MAGNETIC DNA EXTRACTION KIT FOR PLANTS
Background of the Invention The present invention generally relates to the field of methods and kits for the extraction of DNA and specifically to methods and kits for the extraction of DNA from plants.
Methods for extracting DNA from plants include:
The Dellaporta method. Dellaporta. S.L., et al. 1983. A plant DNA
minipreparation: Version II. Plan ~lol Biol Rep 1:19-21, where DNA is precipitated to produce a very crude preparation. with a lot of the ethanol insoluble contaminants present in the solution. A variant of the Dellaporta method introduces an additional step of purifying the DNA via ultracentrifugation through a cesium chloride (CsCI) gradient for several hours or overnight. Richards, E., et al. 1994. Current Protocols ij~ Molecular Biology, Suppl. 27. 2.3.1-2.3.7. Purifying DNA through CsCI gradients is an old established technique that can give very pure DNA, but is time consuming, expensive and results in very low yields.
An alternative DNA extraction method is the cetyltrimethylammonium bromide (CTAB) method. Murry, M.G. et al. 1980. Nucleic Acids Res, 8:4321-4325. CTAB is a cationic detergent. which will form an insoluble complex with the DNA in the presence of low concentrations of salt. such as a 0.~ M sodium chloride (NaCI) solution. In the CTAB method, the original lvsis solution contains about 1.4 M NaCI. The DNA binds with the CTAB when the NaCI
concentration is decreased to 0.5 M. This method is also time consuming and.
because the procedure does not use organic solvents, an additional step needs to be included to clean up the DNA: a final extraction with organic solvents to rid the preparation of polysaccharide and phenolic compounds.
Methods for extraction of DNA from bacterial cells are more well established. Usually cells are lysed by placing the cells in an alkaline environment. Plasmid DNA is separated from bacterial chromosomal DNA by precipitating the chromosomal DNA and leaving the plasmid DNA in the supernatant solution. As described in U.S. Patent No. x,665,544 to Reeve, et al, after the chromosomal DNA is separated from the plasmid DNA, magnetic beads are introduced into the plasmid containing supernatant. The plasmid DNA binds to the beads, and the plasmid DNA is separated from the contaminates in the supernatant. U.S. Patent No. 4,935,342 to Seligson. et al., discloses a general method for the isolation and purification of nucleic acids, based on the binding to and elution from anion exchange columns, including the use of sequential columns containing weak and strong anion exchange materials. U.S. Patent No.
x,705,628 to Hawkins, discloses the use of magnetic microparticles with a coating including functional groups, specifically carboxyl or negatively charged groups, for the purification and isolation of DNA by binding and elution.
International Application WO 96/18731 by Deggerdal, et al., discloses a method for the isolation of nucleic acids by the binding to and elution from a solid support. preferably magnetic beads, in the presence of a detergent, preferably an anionic detergent such as SDS or SARCOSYLTM. U.S. Patent No. 5,650,506 to Woodard, et al., discloses the use of glass fiber membranes bearing positive surface charges for DNA purification by binding to and elution from the glass fibers.
A commercially available method for the extraction of DNA from bacterial cells is marketed as ISOLATETM by Annovis, Inc. (catalog number 2-0300-85). The protocol includes the following steps: suspending bacterial cells in a buffer (pH 8.0)., lysing the cells by mixing the suspension with an alkaline -detergent solution (0.2 M NaOH, 1% SDS), neutralizing the alkali and precipitating the chromosomal DNA with 3.0 M Potassium Acetate, pH 5.~, centrifuging the mixture to sediment the precipitated protein, cell debris and denatured chromosomal DNA, mixing the supernatant containing the plasmid DNA with a suspension containing magnetic beads, binding the DNA to the beads via an anionic interaction, eluting the DNA from the beads, and adjusting the salt concentration to 0.3. adding ethanol to render the plasmid DNA
insoluble and effecting the precipitation of plasmid DNA from the liquid phase. This procedure does not work well with genomic plant DNA, however.
i __ 06-06-2001 )1 6:40PM HOLLAND~'~& KNIGHT ' ; ~. . , N0. 076-° °
. . US 000010834 '# ~~% v a:~ '~.' ..': i w0 00163362 ' ' PCT/US00/10834 , . ,. .w ' ~ ~ : .~; ,.
' ~ ~ It is therefore an object of the present invention to provide a method of extracting and purifying DNA from plants with a high degree of purity and integrity in a short period of time. : ~ , It is also an object of the present invention to provide a kit with . . . v ~~ ;~ ; ;.~ :... ,. . reagents for rapidly and inexpensively extracting and purifying DNA &vm plants_ Brief Summary of the Invention . . ' ., . ,.. ; ~ ': v '%- i . ,p ;i...~ i ~. , .' . : .~. , , :n 4 A method and kit for the extraction of DNA from plants~is provided, . ' which quicl~ly yields plant DNA with a high level of purity. The method isolates DNA (genomic, chloroplast, and/or mitochondriaLDNA) using immobilized cationic groups, preferably on a chromatographic substrate or more preferably E~ .~ .:, ; n?agnctic beads derivatized with cationic groups such as diethylaminoethyl wt (DEAF) via an anion-exchange interaction. The purified DNA is then eluted . .
;.~, .;.;., ~. .' , i; :... ~ ; . ; , with ions (typically a salt solution). RNA can be removed by digestion with ' ~ ~ ~ .
RNAse.
1: . ~ . : ..
Detailed Description of the Invention ,, -i -; , ~ ;.. .: F , , ;The following terms are used herein: ~ .
' ,_ . .
1 ~ ' ' anionic- negatively charged , cationic - positively charged ' '. . ~~:~ !' .~ ' 'y . . .' : ' a. .
f,~~..'',~
i nonionic - non charged Zwitterionic - same number of positive, and negative charges on opposite ends so total compound is neutial.
The method described herein can be used with any plant mateiial and has ,.j; J !t .:~ :~ ~ '1~ . : 5 ~ , n , ._ ' .
'~~been demonstrated to be efficacious with the following representative types of plants: arabdopsis seedlings, barley embryos, tobacco leaves, tomato'leaves, '' soybean hypocotylis and cultured cells, white beans hypocotylis and roots, young and old pint needles.
,. , .
The process generally includes the steps of grinding plant material to make a tissue extract, lysin,g the plant cells, removing cell debris, and binding the ~~ ' ~ a s ~ ~~~ vplant DNA to an immobilized or insoluble material such as magnetic beads, where it is separated into pure form. ~.~ i;. ~~ ' . , ;~ .
f. 3 .
' ~ ~ ~ '° a :~ . ~ ~w ' ~ ' AMENDED SHEET
~:i.::. .. . .:
v Emvfan~gsieit 6.Juni 23:~~
06-06-2001 . ..
,1UN'' 6. ZUUI w b:~40PM ~ HOLLAND & KNIGHT N0. 0576~P. ~S 000010834 ~ . , .Tjj, :L,~~,.~ '' : . ~. ' " ), .'r ' wo oo~s336a YCT/USOO/I0834 .
In a preferred method, plant material is first ground to a powder in liquid ' ' '' '1 :~ t ~' -. , : nitrogen and then incubated in lysis buffer (0.1 M
Tris-HCI, pH 8.0, 0.1 M
EDTA, pH 8.0, 0.25 M NaCI and I00 mierograrnlml proteinaseiK).in the .
presence of a surfactant or nonionic detergent such as N-laurylsarcosine (SARKOSYL""), TRITON'' or NON117ET~ P-40, which acts to solubilize cell .
components and lysc the cells. Representative detergents are listed in Table I.
.Cell debris~is then removed by centrifugation and the supernatant, which . , :, 'f ,~ ~ 1,. ;.., . ..:, feontains the DNA and other soluble cell components, is collected. The DNA is ~ t ' then bound to an immobilized or insoluble matezial having cationic groups bound; ~~.;~:w ;: ,,; ,;
. , ..:y ~i _ ;.~, thereto, such as magnetic beads derivatized with DfiAE. 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 then the DNA is preferably eluted from the, beads by adding NaCl to a final concentration of 1.0 M.
Alternatively, ,, ~. the DNA could be rEmoved by binding to DEAE chromatographic;mat~eri,al or:
,y ;~ ;, I,_. ;. ,t filler material, which is separated by washing, centrifugation, or other methods known to those skilled in the art.
The method and kit produces DNA of equal purity to CsCI gradient methods at yields that are equal to, yr better than, the prior art, Dellaporta and . , ..., ,, ,t ~ ~ ~ ; _,y CTAB, methods. The specific problems of the Dellaporta method (use of a CsCI
1 ~ '~ ' ~~colunnn), and CTAB method (use of cationic detergents), are avoided by this .
~ ~! ~~'~' method - The method and kit also extracts and purifies the DNA more quiekly~ ~
~ ~ ' ' '-than the CsCI method. The method including the precipitation step takes a total of approximately 2.5 hours; without.the precipitation step it takes less than ~. . ~ , , hours (1 hour and 50 minutes) to extract and purify the pleat DNA:
approximately 1, hour to lyre the cells, 5 minutes to bind the DNA to the ~: .
~~ ~ '~~ ~~- magnetic beads, 5 minutes to wash the beads, 5 minutes to separate the DNA , from the beads, 30 minutes to precipitate the DNA from solution, arid 40~iiiinutes,~~~1 t . ~~ ' '' to collect the DNA by centrifugation. In contrast, the CsCI centrifugation rciluires many hours to purify the DNA. ..
:' s ,~ ~ ,~,. _. .,, j . ~'. , 4 ~'~ ~i: :.
lapy : , Emvfangsteit 6.Juni 23 AMENDED SHEET
The preferred method described herein produces a very pure DNA
preparation via an anion-exchange interaction where DNA is replaced as the binding species on the anion-exchange matrix by chloride ions or other negatively charged ions derived from any of a variety of salts. If a traditional anion-exchange column were used at the point where the beads are introduced, the columns would likely clog, preventing collection of bound DNA, or if the elution was effected with strong acid or base, significant levels of contaminates would co-elute with the DNA. In contrast, since the DNA bound to the beads can be thoroughly mixed with the wash solutions, contaminates are more easily removed than they are in traditional column formats, resulting in a more pure preparation. Moreover, because the DNA bound to the beads is not sheared or compressed, 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 anionic detergent in the form of SDS, while the lysis method for plant genomic DNA uses a nonionic detergent. The type of detergent used affects the remaining steps in each method. Since SDS is anionic (negatively charged, like DNA), it must be removed from the solution in the plasmid procedure before the beads are introduced, otherwise the SDS would compete for binding with the plasmid DNA on the positively charged beads. In the ISOLATETM plasmid extraction system, the SDS is removed from the solution by adding potassium acetate to form an insoluble precipitate with the chromosomal DNA. The aggregate of SDS with bacterial genomic DNA can be easily separated from the soluble plasmid DNA. In contrast, in the plant system, no precipitation is needed prior to the binding step, because the nonionic non-charged detergent used in the methods and kits described herein does not compete with the DNA
for binding sites, since the genomic DNA is the desired binding species for the DEAF groups on the beads. Therefore, the nonionic detergent does not need to be removed, whereas SDS and other anionic detergents are negatively charged and do compete with DNA for anionic binding sites and therefore would need to be removed.
Example 1: Purification of Plant Genomic DNA.
The following steps describe extraction and purification of DNA from plants.
1. Harvest approximately 0.5 grams fresh plant tissue. Rinse tissue with deionized (d.I). water to remove adhering debris and blot dry.
2. Freeze tissue with liquid nitrogen and grind to a fine powder in a mortar and pestle. Transfer to a 30 mL conical tube. Alternatively, fresh tissue may be mechanically homogenized in cold lysis buffer at 4°C.
Background of the Invention The present invention generally relates to the field of methods and kits for the extraction of DNA and specifically to methods and kits for the extraction of DNA from plants.
Methods for extracting DNA from plants include:
The Dellaporta method. Dellaporta. S.L., et al. 1983. A plant DNA
minipreparation: Version II. Plan ~lol Biol Rep 1:19-21, where DNA is precipitated to produce a very crude preparation. with a lot of the ethanol insoluble contaminants present in the solution. A variant of the Dellaporta method introduces an additional step of purifying the DNA via ultracentrifugation through a cesium chloride (CsCI) gradient for several hours or overnight. Richards, E., et al. 1994. Current Protocols ij~ Molecular Biology, Suppl. 27. 2.3.1-2.3.7. Purifying DNA through CsCI gradients is an old established technique that can give very pure DNA, but is time consuming, expensive and results in very low yields.
An alternative DNA extraction method is the cetyltrimethylammonium bromide (CTAB) method. Murry, M.G. et al. 1980. Nucleic Acids Res, 8:4321-4325. CTAB is a cationic detergent. which will form an insoluble complex with the DNA in the presence of low concentrations of salt. such as a 0.~ M sodium chloride (NaCI) solution. In the CTAB method, the original lvsis solution contains about 1.4 M NaCI. The DNA binds with the CTAB when the NaCI
concentration is decreased to 0.5 M. This method is also time consuming and.
because the procedure does not use organic solvents, an additional step needs to be included to clean up the DNA: a final extraction with organic solvents to rid the preparation of polysaccharide and phenolic compounds.
Methods for extraction of DNA from bacterial cells are more well established. Usually cells are lysed by placing the cells in an alkaline environment. Plasmid DNA is separated from bacterial chromosomal DNA by precipitating the chromosomal DNA and leaving the plasmid DNA in the supernatant solution. As described in U.S. Patent No. x,665,544 to Reeve, et al, after the chromosomal DNA is separated from the plasmid DNA, magnetic beads are introduced into the plasmid containing supernatant. The plasmid DNA binds to the beads, and the plasmid DNA is separated from the contaminates in the supernatant. U.S. Patent No. 4,935,342 to Seligson. et al., discloses a general method for the isolation and purification of nucleic acids, based on the binding to and elution from anion exchange columns, including the use of sequential columns containing weak and strong anion exchange materials. U.S. Patent No.
x,705,628 to Hawkins, discloses the use of magnetic microparticles with a coating including functional groups, specifically carboxyl or negatively charged groups, for the purification and isolation of DNA by binding and elution.
International Application WO 96/18731 by Deggerdal, et al., discloses a method for the isolation of nucleic acids by the binding to and elution from a solid support. preferably magnetic beads, in the presence of a detergent, preferably an anionic detergent such as SDS or SARCOSYLTM. U.S. Patent No. 5,650,506 to Woodard, et al., discloses the use of glass fiber membranes bearing positive surface charges for DNA purification by binding to and elution from the glass fibers.
A commercially available method for the extraction of DNA from bacterial cells is marketed as ISOLATETM by Annovis, Inc. (catalog number 2-0300-85). The protocol includes the following steps: suspending bacterial cells in a buffer (pH 8.0)., lysing the cells by mixing the suspension with an alkaline -detergent solution (0.2 M NaOH, 1% SDS), neutralizing the alkali and precipitating the chromosomal DNA with 3.0 M Potassium Acetate, pH 5.~, centrifuging the mixture to sediment the precipitated protein, cell debris and denatured chromosomal DNA, mixing the supernatant containing the plasmid DNA with a suspension containing magnetic beads, binding the DNA to the beads via an anionic interaction, eluting the DNA from the beads, and adjusting the salt concentration to 0.3. adding ethanol to render the plasmid DNA
insoluble and effecting the precipitation of plasmid DNA from the liquid phase. This procedure does not work well with genomic plant DNA, however.
i __ 06-06-2001 )1 6:40PM HOLLAND~'~& KNIGHT ' ; ~. . , N0. 076-° °
. . US 000010834 '# ~~% v a:~ '~.' ..': i w0 00163362 ' ' PCT/US00/10834 , . ,. .w ' ~ ~ : .~; ,.
' ~ ~ It is therefore an object of the present invention to provide a method of extracting and purifying DNA from plants with a high degree of purity and integrity in a short period of time. : ~ , It is also an object of the present invention to provide a kit with . . . v ~~ ;~ ; ;.~ :... ,. . reagents for rapidly and inexpensively extracting and purifying DNA &vm plants_ Brief Summary of the Invention . . ' ., . ,.. ; ~ ': v '%- i . ,p ;i...~ i ~. , .' . : .~. , , :n 4 A method and kit for the extraction of DNA from plants~is provided, . ' which quicl~ly yields plant DNA with a high level of purity. The method isolates DNA (genomic, chloroplast, and/or mitochondriaLDNA) using immobilized cationic groups, preferably on a chromatographic substrate or more preferably E~ .~ .:, ; n?agnctic beads derivatized with cationic groups such as diethylaminoethyl wt (DEAF) via an anion-exchange interaction. The purified DNA is then eluted . .
;.~, .;.;., ~. .' , i; :... ~ ; . ; , with ions (typically a salt solution). RNA can be removed by digestion with ' ~ ~ ~ .
RNAse.
1: . ~ . : ..
Detailed Description of the Invention ,, -i -; , ~ ;.. .: F , , ;The following terms are used herein: ~ .
' ,_ . .
1 ~ ' ' anionic- negatively charged , cationic - positively charged ' '. . ~~:~ !' .~ ' 'y . . .' : ' a. .
f,~~..'',~
i nonionic - non charged Zwitterionic - same number of positive, and negative charges on opposite ends so total compound is neutial.
The method described herein can be used with any plant mateiial and has ,.j; J !t .:~ :~ ~ '1~ . : 5 ~ , n , ._ ' .
'~~been demonstrated to be efficacious with the following representative types of plants: arabdopsis seedlings, barley embryos, tobacco leaves, tomato'leaves, '' soybean hypocotylis and cultured cells, white beans hypocotylis and roots, young and old pint needles.
,. , .
The process generally includes the steps of grinding plant material to make a tissue extract, lysin,g the plant cells, removing cell debris, and binding the ~~ ' ~ a s ~ ~~~ vplant DNA to an immobilized or insoluble material such as magnetic beads, where it is separated into pure form. ~.~ i;. ~~ ' . , ;~ .
f. 3 .
' ~ ~ ~ '° a :~ . ~ ~w ' ~ ' AMENDED SHEET
~:i.::. .. . .:
v Emvfan~gsieit 6.Juni 23:~~
06-06-2001 . ..
,1UN'' 6. ZUUI w b:~40PM ~ HOLLAND & KNIGHT N0. 0576~P. ~S 000010834 ~ . , .Tjj, :L,~~,.~ '' : . ~. ' " ), .'r ' wo oo~s336a YCT/USOO/I0834 .
In a preferred method, plant material is first ground to a powder in liquid ' ' '' '1 :~ t ~' -. , : nitrogen and then incubated in lysis buffer (0.1 M
Tris-HCI, pH 8.0, 0.1 M
EDTA, pH 8.0, 0.25 M NaCI and I00 mierograrnlml proteinaseiK).in the .
presence of a surfactant or nonionic detergent such as N-laurylsarcosine (SARKOSYL""), TRITON'' or NON117ET~ P-40, which acts to solubilize cell .
components and lysc the cells. Representative detergents are listed in Table I.
.Cell debris~is then removed by centrifugation and the supernatant, which . , :, 'f ,~ ~ 1,. ;.., . ..:, feontains the DNA and other soluble cell components, is collected. The DNA is ~ t ' then bound to an immobilized or insoluble matezial having cationic groups bound; ~~.;~:w ;: ,,; ,;
. , ..:y ~i _ ;.~, thereto, such as magnetic beads derivatized with DfiAE. 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 then the DNA is preferably eluted from the, beads by adding NaCl to a final concentration of 1.0 M.
Alternatively, ,, ~. the DNA could be rEmoved by binding to DEAE chromatographic;mat~eri,al or:
,y ;~ ;, I,_. ;. ,t filler material, which is separated by washing, centrifugation, or other methods known to those skilled in the art.
The method and kit produces DNA of equal purity to CsCI gradient methods at yields that are equal to, yr better than, the prior art, Dellaporta and . , ..., ,, ,t ~ ~ ~ ; _,y CTAB, methods. The specific problems of the Dellaporta method (use of a CsCI
1 ~ '~ ' ~~colunnn), and CTAB method (use of cationic detergents), are avoided by this .
~ ~! ~~'~' method - The method and kit also extracts and purifies the DNA more quiekly~ ~
~ ~ ' ' '-than the CsCI method. The method including the precipitation step takes a total of approximately 2.5 hours; without.the precipitation step it takes less than ~. . ~ , , hours (1 hour and 50 minutes) to extract and purify the pleat DNA:
approximately 1, hour to lyre the cells, 5 minutes to bind the DNA to the ~: .
~~ ~ '~~ ~~- magnetic beads, 5 minutes to wash the beads, 5 minutes to separate the DNA , from the beads, 30 minutes to precipitate the DNA from solution, arid 40~iiiinutes,~~~1 t . ~~ ' '' to collect the DNA by centrifugation. In contrast, the CsCI centrifugation rciluires many hours to purify the DNA. ..
:' s ,~ ~ ,~,. _. .,, j . ~'. , 4 ~'~ ~i: :.
lapy : , Emvfangsteit 6.Juni 23 AMENDED SHEET
The preferred method described herein produces a very pure DNA
preparation via an anion-exchange interaction where DNA is replaced as the binding species on the anion-exchange matrix by chloride ions or other negatively charged ions derived from any of a variety of salts. If a traditional anion-exchange column were used at the point where the beads are introduced, the columns would likely clog, preventing collection of bound DNA, or if the elution was effected with strong acid or base, significant levels of contaminates would co-elute with the DNA. In contrast, since the DNA bound to the beads can be thoroughly mixed with the wash solutions, contaminates are more easily removed than they are in traditional column formats, resulting in a more pure preparation. Moreover, because the DNA bound to the beads is not sheared or compressed, 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 anionic detergent in the form of SDS, while the lysis method for plant genomic DNA uses a nonionic detergent. The type of detergent used affects the remaining steps in each method. Since SDS is anionic (negatively charged, like DNA), it must be removed from the solution in the plasmid procedure before the beads are introduced, otherwise the SDS would compete for binding with the plasmid DNA on the positively charged beads. In the ISOLATETM plasmid extraction system, the SDS is removed from the solution by adding potassium acetate to form an insoluble precipitate with the chromosomal DNA. The aggregate of SDS with bacterial genomic DNA can be easily separated from the soluble plasmid DNA. In contrast, in the plant system, no precipitation is needed prior to the binding step, because the nonionic non-charged detergent used in the methods and kits described herein does not compete with the DNA
for binding sites, since the genomic DNA is the desired binding species for the DEAF groups on the beads. Therefore, the nonionic detergent does not need to be removed, whereas SDS and other anionic detergents are negatively charged and do compete with DNA for anionic binding sites and therefore would need to be removed.
Example 1: Purification of Plant Genomic DNA.
The following steps describe extraction and purification of DNA from plants.
1. Harvest approximately 0.5 grams fresh plant tissue. Rinse tissue with deionized (d.I). water to remove adhering debris and blot dry.
2. Freeze tissue with liquid nitrogen and grind to a fine powder in a mortar and pestle. Transfer to a 30 mL conical tube. Alternatively, fresh tissue may be mechanically homogenized in cold lysis buffer at 4°C.
3: Add 5-20 ml of lysis buffer/gram of starting material (0.5 to 0.01 M Tris-HCI, pH7.0-8.0,0.001-O.SMEDTA,pH7.0-8.0,0.05-0.4MNaCland50-X00 qg/ml proteinase K) to the frozen powder and mix well.
4. Add N-lauroylsarcosine at a final concentration of between 0.1 % and 10%.
If desired, 25 - 200 qg of RNase A can be added at this point. Alternatively, the final resuspended pellet can be treated with RNase. Incubate 30 minutes to 1 hour at 50-60°C.
If desired, 25 - 200 qg of RNase A can be added at this point. Alternatively, the final resuspended pellet can be treated with RNase. Incubate 30 minutes to 1 hour at 50-60°C.
5. Centrifuge lysate 10 min at greater than SOOOx g at 4°C to pellet debris.
Remove the supernatant to a fresh tube and filter, if necessary, to remove undigested debris.
Remove the supernatant to a fresh tube and filter, if necessary, to remove undigested debris.
6. Add from 1 - 30 mL of magnetic beads (40 % v/v suspension) to supernatant.
Gently mix the bead-supernatant suspension by inversion or mechanical mixer at room temperature for at least 1 minute.
Gently mix the bead-supernatant suspension by inversion or mechanical mixer at room temperature for at least 1 minute.
7. Immobilize the magnetic beads in the magnetic separator and remove supernatant.
8. Add between 5 and 20 ml of wash buffer, cap and remove the tube from the stand and gently mix for 5-10 minutes at room temperature.
9. Immobilize the magnetic beads in the magnetic separator and remove supernatant. Repeat wash, if supernatant is substantially pigmented.
10. Add between ~ and 10 ml of elution buffer (0.7 - 1.5 M NaCI, 0.5 to 0.01 M
Tris-HCI, pH 7.0 - 8.0, 0.001-0.5 M EDTA. pH 7.0 - 8.0) to the magnetic beads and mix for ~ - 10 minutes at room temperature.
Tris-HCI, pH 7.0 - 8.0, 0.001-0.5 M EDTA. pH 7.0 - 8.0) to the magnetic beads and mix for ~ - 10 minutes at room temperature.
11. Immobilize the magnetic beads in the magnetic separator and transfer supernatant to a fresh tube.
12. Add 1.0 to 2.5 volumes of cold isopropanol. Mix well and incubate at -20°C for at least 10 min.
13. Collect DNA by centrifugation at 10,000 - 20,000 x g for at least 15 min at 4°C. Wash pellet with 70% cold ethanol and re-centrifuge at 10,000 -20,000 x g for at least 2 min at 4°C. Allow the pellet to air-dry at room temperature and then dissolve in desired buffer.
It is understood that the disclosed method and kit is not limited to the particular methodology. protocols, and reagents described. as these may vary.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Table I: Representative Detergents which can be used in the Plant DNA Extraction Method (available from Sigma Chemical Co) Anionic Detergents Aerosol 22 Glycodeoxycholic Acid Aerosol-OT 1-Heptanesulfonic Acid Salts of: 1-Hexanesulfonic Acid Alginic Acid N-Lauroylsarcosine Caprylic Acid Lauryl Sulfate (Dodecyl sulfate) Cholic Acid 1-Nonanesulfonic Acid 1-Decanesulfonic Acid 1-Octanesulfonic Acid Dehydrocholic Acid 1-Pentanesulfonic Acid Deoxycholic Acid Taurocholic Acid Dioctyl Sulfosuccinate Taurodeoxycholic Acid 1-Dodecanesulfonic Acid Niaproof (formerly Tergitol) Glycocholic Acid Cationic Detergents Alkyltrimethylammonium Bromides Benzalkonium Chloride Benzothonium Chloride Benzyldimethyldodecylammonium Bromide Benzyldimethylhexadecylammonium Chloride Benzyldimethyletetradecylammonium Chloride Cetyldimethylethylammonium Bromide Cetylpyridinium Decamethonium Bromide Dimethyldioctadecylammonium Bromide Methvlbenzethonium Chloride Methyltrictylammonium Chloride N, N',N'-Polyoxyethylene(10)-N-tallow-l, 3-diaminopropane Zwitterionic Detergents CHAPS
CHAPSO
N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate N-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate N-Hexadecyl-N, N-dimethyl-3-amonio-1-propanesulfonate N-Octadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate N-Octyl-N, N-dimethyl-3-ammonio-1-propanesulfonate Phosphatidylcholine N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate Table 1 continued.
Nonionic Detergents BIGCHAP n-Nonyl -D-Glucopyranoside Decanoyl-N-methylglucamiden-Nonyl -D-Glucopyranoside n-Decyl -D-GlucopyranosideOctanoyl-N-methylglucamide n-Decyl -D-Glucopyranosiden-Octyl -D-Glucopyranoside n-Decyl -D-Maltopyranosiden-Octyl -D-Glucopyranoside Deoxy-BIGCHAP Octyl -D-Thiogalactopyranoside n-Dodecyl -D-GlucopyranosidePolyoxyethylene Esters n-Dodecyl -D-Maltoside Polyoxyethylene Ethers n-Dodecyl -D-Maltoside Polyoxyethylenesorbitan Esters Heptanoyl-N-methylglucamideSorbitan Esters n-Heptyl -D-GlucopyranosideTergitol N-Heptyl -Thioglucopyranosiden-Tetradecyl -D-Maltoside n-Hexyl -D GlucopyranosideTritons Igepal CA-630 Tyloxapol 1-Monooleoyl-rac-glyceroln-Undecyl -D-Glucopyranoside Nonanoyl-N-methylglucamide
It is understood that the disclosed method and kit is not limited to the particular methodology. protocols, and reagents described. as these may vary.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Table I: Representative Detergents which can be used in the Plant DNA Extraction Method (available from Sigma Chemical Co) Anionic Detergents Aerosol 22 Glycodeoxycholic Acid Aerosol-OT 1-Heptanesulfonic Acid Salts of: 1-Hexanesulfonic Acid Alginic Acid N-Lauroylsarcosine Caprylic Acid Lauryl Sulfate (Dodecyl sulfate) Cholic Acid 1-Nonanesulfonic Acid 1-Decanesulfonic Acid 1-Octanesulfonic Acid Dehydrocholic Acid 1-Pentanesulfonic Acid Deoxycholic Acid Taurocholic Acid Dioctyl Sulfosuccinate Taurodeoxycholic Acid 1-Dodecanesulfonic Acid Niaproof (formerly Tergitol) Glycocholic Acid Cationic Detergents Alkyltrimethylammonium Bromides Benzalkonium Chloride Benzothonium Chloride Benzyldimethyldodecylammonium Bromide Benzyldimethylhexadecylammonium Chloride Benzyldimethyletetradecylammonium Chloride Cetyldimethylethylammonium Bromide Cetylpyridinium Decamethonium Bromide Dimethyldioctadecylammonium Bromide Methvlbenzethonium Chloride Methyltrictylammonium Chloride N, N',N'-Polyoxyethylene(10)-N-tallow-l, 3-diaminopropane Zwitterionic Detergents CHAPS
CHAPSO
N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate N-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate N-Hexadecyl-N, N-dimethyl-3-amonio-1-propanesulfonate N-Octadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate N-Octyl-N, N-dimethyl-3-ammonio-1-propanesulfonate Phosphatidylcholine N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate Table 1 continued.
Nonionic Detergents BIGCHAP n-Nonyl -D-Glucopyranoside Decanoyl-N-methylglucamiden-Nonyl -D-Glucopyranoside n-Decyl -D-GlucopyranosideOctanoyl-N-methylglucamide n-Decyl -D-Glucopyranosiden-Octyl -D-Glucopyranoside n-Decyl -D-Maltopyranosiden-Octyl -D-Glucopyranoside Deoxy-BIGCHAP Octyl -D-Thiogalactopyranoside n-Dodecyl -D-GlucopyranosidePolyoxyethylene Esters n-Dodecyl -D-Maltoside Polyoxyethylene Ethers n-Dodecyl -D-Maltoside Polyoxyethylenesorbitan Esters Heptanoyl-N-methylglucamideSorbitan Esters n-Heptyl -D-GlucopyranosideTergitol N-Heptyl -Thioglucopyranosiden-Tetradecyl -D-Maltoside n-Hexyl -D GlucopyranosideTritons Igepal CA-630 Tyloxapol 1-Monooleoyl-rac-glyceroln-Undecyl -D-Glucopyranoside Nonanoyl-N-methylglucamide
Claims (14)
1. A method for extracting DNA from plant material comprising (a) grinding the plant material to make a plant material extract, (b) lysing the cells of the plant material extract in a lysis buffer solution comprising,0.5 to 0.01 M Tris-HCI, pH 7.0 - 8.0, 0.001- 0.5 M EDTA, pH 7.0 -8.0, 0,05 - 0.4 M NaCI and 50-500 µg/ml proteinase K, (c) removing the plant cell material debris, (d) binding the DNA to positively charged groups wich are immobilized on beads or insoluble, (e) separating the bound DNA from the remaining cellular material, and (f) separating the bound DNA from the positively charged beads by increasing the salt ion concentration of an elution buffer.
2. The method of claim 1 wherein tho lysis buffer comprises a non-ionic detergent.
3. The method of claim 1 wherein the positively charged goups are DEAE.
4. The method of claim 1 wherein the 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 of claim 1 wherein the elution buffer comprises, 0.7 -1.5M NaCI, 0.5 to 0.01 M Tris-HCI, pH 7.0-8.0, 0.001-0.5M EDTA, pH 7.0 - 8.0,
7. The method of claim 1 further comprising adding, RNAse to the lysis buffer to digest the RNA in the plant material or purified DNA.
8. A kit fox use in a method for extracting DNA from plant material comprising (a) a plant material extract made from grinding the plant material, (b) a lysis buffer solution fot lysing the cells in the plant material extract comprising, 0.5 to 0,01 M Tris-HC1, pH 7.0 - 8.0, 0.001- 0.5 M EDTA, pH 7.0 -8.0, 0.05 - 0.4 M NaCI and 50-500 µg/ml proteinase K, (c) positively charged groups which are immobilized on beads or insoluble, and (d) an elution buffer for separating the bound DNA from the positively charged groups comprising, 0.7-1.5M NaCI, 0.5 to 0.01 M Tris-HCL, pH 7.0 - 8.0, 0.001 -0.5M EDTA, pH7.0 - 8Ø
9. The kit of claim 8 wherein the solution to lyse the cells is a non-ionic solution.
10. The kit of claim 8 wherein the positively charged groups are DEAE
immobilized on a chromatrographic substrate or magnetic beads.
immobilized on a chromatrographic substrate or magnetic beads.
11. The kit of claim 8 comprising:
(a) a plant material extract made from grinding the plant material, (b) lysis buffer comprising 0.5 to 0.01 M Tris-HC1, pH 7.0 - 8.0, 0.001 - 0.5 M
EDTA, pH 7Ø- 8.0, 0.05 - 0.4 M NaC1 and 50 - 500 µg/ml proteinase K;
(c) N-lauroylsarcosine;
(d) magnetic beads having DEAE groups on the surface thereof; and (e) elution buffer comprising 0.7 - 1.5 M NaC1, 0.5 to 0.01 M Tris-HC1, pH 7.0 -8.0,0.001-0.5M EDTA,pH7.0-8Ø
(a) a plant material extract made from grinding the plant material, (b) lysis buffer comprising 0.5 to 0.01 M Tris-HC1, pH 7.0 - 8.0, 0.001 - 0.5 M
EDTA, pH 7Ø- 8.0, 0.05 - 0.4 M NaC1 and 50 - 500 µg/ml proteinase K;
(c) N-lauroylsarcosine;
(d) magnetic beads having DEAE groups on the surface thereof; and (e) elution buffer comprising 0.7 - 1.5 M NaC1, 0.5 to 0.01 M Tris-HC1, pH 7.0 -8.0,0.001-0.5M EDTA,pH7.0-8Ø
12. The kit of claim 8 further comprising RNAse in the lysis buffer.
13. The kit of claim 11 further comprising RNAse in the lysis buffer.
14. The method of claim 1 wherein flue lysis buffer contains N-lauroylsarkosine.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US13035399P | 1999-04-21 | 1999-04-21 | |
| US60/130,353 | 1999-04-21 | ||
| PCT/US2000/010834 WO2000063362A1 (en) | 1999-04-21 | 2000-04-21 | Magnetic dna extraction kit for plants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2370656A1 true CA2370656A1 (en) | 2000-10-26 |
Family
ID=22444292
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002370656A Abandoned CA2370656A1 (en) | 1999-04-21 | 2000-04-21 | Magnetic dna extraction kit for plants |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1171584A1 (en) |
| JP (1) | JP2002541839A (en) |
| AU (1) | AU4367500A (en) |
| CA (1) | CA2370656A1 (en) |
| WO (1) | WO2000063362A1 (en) |
Families Citing this family (42)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040121309A1 (en) | 2002-12-06 | 2004-06-24 | Ecker David J. | Methods for rapid detection and identification of bioagents in blood, bodily fluids, and bodily tissues |
| US7226739B2 (en) | 2001-03-02 | 2007-06-05 | Isis Pharmaceuticals, Inc | Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations |
| US20030027135A1 (en) | 2001-03-02 | 2003-02-06 | Ecker David J. | Method for rapid detection and identification of bioagents |
| US7666588B2 (en) | 2001-03-02 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy |
| US8073627B2 (en) | 2001-06-26 | 2011-12-06 | Ibis Biosciences, Inc. | System for indentification of pathogens |
| US7217510B2 (en) | 2001-06-26 | 2007-05-15 | Isis Pharmaceuticals, Inc. | Methods for providing bacterial bioagent characterizing information |
| US7208271B2 (en) | 2001-11-28 | 2007-04-24 | Applera Corporation | Compositions and methods of selective nucleic acid isolation |
| CA2508726A1 (en) | 2002-12-06 | 2004-07-22 | Isis Pharmaceuticals, Inc. | Methods for rapid identification of pathogens in humans and animals |
| US8057993B2 (en) | 2003-04-26 | 2011-11-15 | Ibis Biosciences, Inc. | Methods for identification of coronaviruses |
| US7964343B2 (en) * | 2003-05-13 | 2011-06-21 | Ibis Biosciences, Inc. | Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US8158354B2 (en) * | 2003-05-13 | 2012-04-17 | Ibis Biosciences, Inc. | Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
| US20050009036A1 (en) * | 2003-07-11 | 2005-01-13 | Applera Corporation | Methods and kits for obtaining nucleic acid from biological samples |
| US8546082B2 (en) | 2003-09-11 | 2013-10-01 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US8097416B2 (en) | 2003-09-11 | 2012-01-17 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
| US7956175B2 (en) | 2003-09-11 | 2011-06-07 | Ibis Biosciences, Inc. | Compositions for use in identification of bacteria |
| US7666592B2 (en) | 2004-02-18 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for concurrent identification and quantification of an unknown bioagent |
| WO2005117270A2 (en) | 2004-05-24 | 2005-12-08 | Isis Pharmaceuticals, Inc. | Mass spectrometry with selective ion filtration by digital thresholding |
| US20050266411A1 (en) | 2004-05-25 | 2005-12-01 | Hofstadler Steven A | Methods for rapid forensic analysis of mitochondrial DNA |
| US7811753B2 (en) | 2004-07-14 | 2010-10-12 | Ibis Biosciences, Inc. | Methods for repairing degraded DNA |
| WO2006135400A2 (en) | 2004-08-24 | 2006-12-21 | Isis Pharmaceuticals, Inc. | Methods for rapid identification of recombinant organisms |
| CA2579804C (en) | 2004-09-16 | 2013-12-10 | Cropdesign N.V. | Root evaluation |
| KR100601972B1 (en) | 2004-11-03 | 2006-07-18 | 삼성전자주식회사 | Apparatus and method for the purification of nucleic acids by phase separation using Laser and beads |
| WO2006094238A2 (en) | 2005-03-03 | 2006-09-08 | Isis Pharmaceuticals, Inc. | Compositions for use in identification of adventitious viruses |
| US8084207B2 (en) | 2005-03-03 | 2011-12-27 | Ibis Bioscience, Inc. | Compositions for use in identification of papillomavirus |
| CA2616281C (en) | 2005-07-21 | 2014-04-22 | Isis Pharmaceuticals, Inc. | Methods for rapid identification and quantitation of mitochondrial dna variants |
| AU2007353877B2 (en) | 2006-09-14 | 2012-07-19 | Ibis Biosciences, Inc. | Targeted whole genome amplification method for identification of pathogens |
| WO2008104002A2 (en) | 2007-02-23 | 2008-08-28 | Ibis Biosciences, Inc. | Methods for rapid forensic dna analysis |
| US9598724B2 (en) | 2007-06-01 | 2017-03-21 | Ibis Biosciences, Inc. | Methods and compositions for multiple displacement amplification of nucleic acids |
| US8148163B2 (en) | 2008-09-16 | 2012-04-03 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
| EP2337441B1 (en) | 2008-09-16 | 2016-08-10 | BASF Plant Science GmbH | Method for improved plant breeding |
| WO2010033599A2 (en) | 2008-09-16 | 2010-03-25 | Ibis Biosciences, Inc. | Mixing cartridges, mixing stations, and related kits, systems, and methods |
| US8534447B2 (en) | 2008-09-16 | 2013-09-17 | Ibis Biosciences, Inc. | Microplate handling systems and related computer program products and methods |
| EP2396803A4 (en) | 2009-02-12 | 2016-10-26 | Ibis Biosciences Inc | Ionization probe assemblies |
| EP2454000A4 (en) | 2009-07-17 | 2016-08-10 | Ibis Biosciences Inc | Systems for bioagent identification |
| US8950604B2 (en) | 2009-07-17 | 2015-02-10 | Ibis Biosciences, Inc. | Lift and mount apparatus |
| EP2957641B1 (en) | 2009-10-15 | 2017-05-17 | Ibis Biosciences, Inc. | Multiple displacement amplification |
| CN102533731A (en) * | 2012-01-19 | 2012-07-04 | 西北农林科技大学 | Extraction kit and extraction method for tomato genome total DNA (Deoxyribonucleic Acid) |
| CN102618532A (en) * | 2012-05-02 | 2012-08-01 | 易春 | Kit for extracting genome DNA (Deoxyribose Nucleic Acid) from plant leaves based on paramagnetic particle method and extracting method thereof |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
| CN110452905B (en) * | 2019-08-22 | 2023-06-02 | 云南省烟草农业科学研究院 | Extraction method for improving tobacco DNA precipitation efficiency and application thereof |
| CN110669759A (en) * | 2019-10-28 | 2020-01-10 | 北京百迈客生物科技有限公司 | Method for extracting fungus high-purity long-fragment genome DNA (deoxyribonucleic acid) suitable for nanopore sequencing |
| CN112921028A (en) * | 2019-12-06 | 2021-06-08 | 深圳华大基因科技服务有限公司 | DNA purification method, genomic DNA extraction method, sequencing method and kit |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4997932A (en) * | 1989-11-13 | 1991-03-05 | Boehringer Mannheim Corporation | Method and kit for purifying nucleic acids |
| GB9425138D0 (en) * | 1994-12-12 | 1995-02-08 | Dynal As | Isolation of nucleic acid |
| US5981235A (en) * | 1996-07-29 | 1999-11-09 | Promega Corporation | Methods for isolating nucleic acids using alkaline protease |
| KR20010032806A (en) * | 1997-12-06 | 2001-04-25 | 매튜 존 베이커 | Isolation of nucleic acids |
-
2000
- 2000-04-21 AU AU43675/00A patent/AU4367500A/en not_active Abandoned
- 2000-04-21 CA CA002370656A patent/CA2370656A1/en not_active Abandoned
- 2000-04-21 JP JP2000612441A patent/JP2002541839A/en not_active Withdrawn
- 2000-04-21 WO PCT/US2000/010834 patent/WO2000063362A1/en active Search and Examination
- 2000-04-21 EP EP00923575A patent/EP1171584A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP1171584A1 (en) | 2002-01-16 |
| WO2000063362A1 (en) | 2000-10-26 |
| AU4367500A (en) | 2000-11-02 |
| JP2002541839A (en) | 2002-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2370656A1 (en) | Magnetic dna extraction kit for plants | |
| US7931920B2 (en) | Method for the isolation of nucleic acids from any starting material | |
| DE4321904B4 (en) | Method for chromatographic purification and separation of nucleic acid mixtures | |
| US5783686A (en) | Method for purifying nucleic acids from heterogenous mixtures | |
| WO1999040098A1 (en) | Method for isolating and purifying nucleic acids | |
| US6797476B2 (en) | Process for the scaleable purification of plasmid DNA | |
| EP1851313A1 (en) | Method for isolating nucleic acids, the nucleic acids being immobilised on a matrix at an increased temperature | |
| US8029991B2 (en) | Method and formulation for the extraction of nucleic acids from any complex starting materials | |
| EP1560926B2 (en) | Novel buffer formulations for isolating, purifying and recovering long-chain and short-chain nucleic acids | |
| CA2393479C (en) | Process for the scaleable purification of plasmid dna | |
| WO2005045030A1 (en) | A rapid and low cost method for isolating nucleic acid | |
| EP3228710B1 (en) | Purification of nucleic acids from a sample comprising nucleic acids and endotoxin | |
| DE10253351A1 (en) | Isolation of nucleic acid by binding to solid phase, useful e.g. in forensic or diagnostic medicine, uses binding buffer containing monovalent and multivalent ions, eliminates need for alcohol in wash buffer | |
| EP2157181A1 (en) | Method for isolating nucleic acids and test kit | |
| CN116676303A (en) | Soil RNA extraction kit free of water saturated phenol and chloroform, extraction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |