CA2237329A1 - Extraction system and method - Google Patents
Extraction system and method Download PDFInfo
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- CA2237329A1 CA2237329A1 CA002237329A CA2237329A CA2237329A1 CA 2237329 A1 CA2237329 A1 CA 2237329A1 CA 002237329 A CA002237329 A CA 002237329A CA 2237329 A CA2237329 A CA 2237329A CA 2237329 A1 CA2237329 A1 CA 2237329A1
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- hollow tube
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- tube assembly
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Pathology (AREA)
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- Immunology (AREA)
- Cell Biology (AREA)
- Hydrology & Water Resources (AREA)
- Sustainable Development (AREA)
- Crystallography & Structural Chemistry (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A novel system for extracting a substance of interest, e.g., a chemical or biological substance, from a biological sample involves a hollow tube apparatus for receiving the sample, a compressing means for expelling the substance of interest from the sample, and a collection means for collecting the expressed substance. The invention also includes methods of use for the described system and kits containing components of the system.
Description
WO 97/18293 PCT~US96/17951 EXTRACTION SYSTEM AND METHOD
Cross-Reference to a Related Appli~tinn This is a c ,~ nl;on-in-part of co-pending application Serial No. 60/006,626, filed ~ 5 November 10, 1995.
Field of the Invention The present invention relates to the PYfrActinn of ~ ~ped s~ ln.~rcs from the Sulluulldill$ material in a hiol<~AI satnple and, more particularly, to a system and method fûr 0 CUlll~ illg a hinlclgicAI sample to extract a b~bbin-.~e of interest. ~dtlitinnAlly, the present invention relates to an extraction kit used to practice the present extraction process.
Back~round of the Invention Separation and extraction l~ ~ .es to obtain ~hPmi~AI or binlc.gi~-AI bul,s~lces from a kinl<gicAI sample are commnn p-w~s to many research, ~liA~n,cti~" IL~la~Jc~lLic and cc~ ~ah ~., 'v~s. Fl~i4u~lllly, a ~ e of interest will need to be l~llluv~d from a material matrix in order to fiA~ilit~ se.l~;nn for analysis. For P~Amrl~, plant biology .t;se~u~llers often desire to extract x~ ;... Pc, such as nucleic acids and proteins, found in plant leaves or other tissues. Such sllhstancff~ct extraction of the s~llLJsi n.-.~e The s--hsfAnre may also be physically c~.~.ay~ed by the solid matrix ~e.g. cell walls~, making even lengthy elution periods inA-le~nAte to extract the b~hb~ e from the matrix.
~otnri7Pcl roller devices have long been used to express b'.b.~ 'C such as cell sap from plant tissues. IIv.._~,., this apl!.oacl- is not well-suited when many, small samples need to be al~,l because it is slow and messy and the rol}ers must be cleaned and, in certain ;..x~ c ~ rd between sample runs. Also, it is difficult to collect the expelled ~l,bl A~ ~rc and particularly so to obtain ~ r; Vl;i extraction.
In the extraction of nucleic acids (DNA, RNA) from plant tissues, cnmrlicAtP~ and time c .. x.. ;.. g methods hinder an efficient extraction and purifirAtinn process. For P~ Ample, plant tissues are norrnally physically hnmngPni7p~d in a buffer which sol--hili7Pc the nucleic acid.
~mogP.ni7Atinn is a~mplixhpd by such means as a mortar and pestle, a food grinder or a hnmogPni~r (POLYTRON brand from n~ . ~PrhAnic~Al disruption is often ~1iffirult with very small ~nAntitiPC of plant tissue making it difficult to work with young plants.
After grinding, nucleic acids are then ~ led from the tissue slurry in a process that may involve multiple organic solvent P~innC ~chloroform, phenol, etc.) and centrifi-~Atinnc This is not only CA 02237329 1998-05-ll W O 97/18293 PCT~US96/17951 tcdious work but is also time c~ .;.-g Cross cn~ ;nn is a major concern with ml~r,h~mir,~l grinding as analytical f~chniq~ , e.g. PCR, become more ~clL7ilive Bnef Summary of the Invention The ~;ALIa~;Liull system ofthe present invention allows the efficient extraction of a snhst~nre of interest from a bil logir~l sample in minimal time, and with a minimal amount of eluant buffer.
Mess and system c ~ nn are ~ -;~ P~l by c-~ ;..;..g the sample and eluant buffer in a hollow tubular assembly, such as a plastic straw, such that tl~e sample never comes in contact with certain other c-....l.u.~ of the extraction system that are adv~nt~g.o~ lely kept from be~,....-;-.g c~ ln~
In ane aspect of the present invention the system includes a hollow tube assembly adapted to contain a sample and a means for cnllP,cfing the ~ e of interest. A Cc,lL~ g means operates in c-~. . .k; ~ r I ;nn with the hollow tube assembly to deform the region of the hollow tube assembly c.~ ;. .;. .g the sample, thereby cc)lllp~ ~g the sample and ~.Ypellin~ at least a portion of 1~ the ~ n. .. ~ of interest L~ L Ulll which is cc llecte~l Once collecte~1 the ~ of interest may be further purified and analyzed employing standard ~ ica~ion and analylical terhniqll~e A further aspect of the present invention involves a method of using the e~trFIrtinn system to separate the ~,..l.s~ .cG of interest from the sample. The extraction method is initiated by plVV' " g a hollow tube assembly havirlg a flefr- -- ' ' ~ region forc- ....n;~ the sample. The sample is placed within the ~.r~ ble region of the hollow tube assembly. ColL~ i.,A,iull means are then used to deform the hollow tube and ~ the sarnple: ~ ~ ' therein. The ~" ~1 .,e, 1~ ~r c of interest is then s~ nl~1 from the iiquid expelled from the sample, purified, and analyzed ernploying known ~iLcalion and analytical te~lmiques Of particular interest in the practice of the present illv~ulioll, an extraction kit is ~r~al~l and sold to those i ll~lc.,led in ~AIla iLillg ."~ 3 ~rP s of interest (DNA, RNA, proteins, etc.) from bifl~L, ' sarnples such as plants. The kit c. ~ ; all of the m~tf .ri5l1c, with the RY~f~ptinn of the co.ll~les,;on means, to enable one to carry out an efficient ~"~ha_liVIl process acculding to the present .. ~ iulL The kit will typically include a hûllow tube assembly (straw), a filter paper to hold the sample, a coll~,ti~n tube with packing and buffer solnti~ns to ~ta~ili7~?~ extract and/or purify the ~. .l .si ~ e of interest.
Rri~.f J~escription of the Drawings Figures lA-lD are a ~ f .~ n of the process of placing a bi~l~gic~l sample (plant leaf) Ul filter paper, and insertion thereof into a hollow tube a~ bly;
Figure 2 is a 1~ '71'~ ti~n of one embodiment of the system of the present invention;
Figures 3A and 3B show embodunents of the c~l1Rction means having filter discs disposed therein, FiglFire 4 is a photograph depicting the pPrfnrrn~nce of a method of the present invention.
~ 5 Figure S is a photograph of a 96 well pl~fnrm c~ .;.l;.. g two cn11ectinn tubes.
Figure 6 is a phntograrh showing an empty cnllPcti- n tube (right), a C~ llec.tir~n tube fully loaded with a filter disc (left), and the Cn~ U ~- ~~ ; of a filter, i.e., filter paper sandwiched in bctween two discs of porous polyethylene.
Figure 7 is a p1.~.1.~ ,.i.1~ of a hollow tube assembly, a filter paper to hold the tissue sample and a co11~cti. n tube.
Figure 8 is a photograph showing an Pmhor1imRnt of a conical hollow tube assembly according to the subject invention.
Detailed Disclosure of the Invention The present invention involves an a~pdLaLu~ and methods of use for co11ectin~ a ~.h~ e of interest c- ...1 i.;..~d in a biok~r~1 sample. The a~l~aLus of the subject invention c~....l.. ;~f c; a hollow tube assembly (h~ ~arL~l referred to as "hollow tube") for receiving a sample in a d~f()rm ~hle region thereof, a C~1~)1~i'7 7illg means for culn~n~ g said region having the sample thOEein, and a means for collPcting the ~ ~bs~ e of interest for b~ se~ -l p-lrifi~ti(m or physical, ~ ,- 1, or chPm~ l analysis. The cu.ll~l~s~ion of the sample by the Cu~ iug means bursts or disrupts the sample matrix (e.g., cells) thereby releasing the ~..l.~l,...- e of intOEest (e.g., cell c~.-l. .a.;) from the matrix into a buffer ~lRci~Pd to stabilize the specific b~l)71i~ac. of interest.
C~ ,;~. of the sample can furlhOE express the liquid c- .. ~ the ~ e of intOEest out of the hollow tube and into a cn11Prti~n means (l~ aL~I "c< 1lpctir n tube") for ~d-liticm~
of the "~ e of interest. The purified ~b~ e of interest can then be analyzed employing standard analytical tca]-~
The claimed invention includes the system, mt?i~ning the physical Cl~ "" ..1~; in operable n (;".1.,~ an c~a~,Liull kit employed to practice the present invention), and the method of using the system to extract, purify and analyze a ~ e of interest from a sample. While the ~.. 1.o l;.". .,1.; ~i~c~ . ;hed herein include a roller ~alaLus as a CU~ illg means for CC~ .. lg the sample, other equivalent ~- F~ ..c for dPfnrming the f4 rc - ...ilhle region of the hollow tube, and thOEeby Culll~ hlg the sample c~ Pd therein, are c~ tR I such as, for P~rnmple7 a press, or other device known to the ordinarily skilled artisan. Likewise, fimrtinnil11y e~uival~,.lL
hald~e can be g~h~ d for the hollow tube a.,~mbly and sample cnllRr,tinn tube.
CA 02237329 1998-05-ll W O 97/18293 PCT~US96/17951 The present invention will be found usefi~l in many sitllAti~nc whe}e it is desired to extract a substQn~ e. of inte}est from a bielcgl.~Al material matrix in order to fAeilitAf~ suhseq~l.nt manipulation for analysis. For t.~Amrle, 7~ es such as nucleic acids, proteins, and other I) o~ P~ c found in plant tissues (e~g~ leaves) can be ~ cc~scrlllly s~ t~d from the plant tissues by ~u~lvy--lg the illvc-l~tion~ In a~dition, nucleic acids, proteins, viruses, bacteria, carboLy~aL~;s and hi.~ it'.AIc found in plant and animal tissues or other bi.~l. gicQI mQtrirçc can be s~ d and ~ -1. a~;L~I by rrQrtit~ing the present ~~lvellLioll. Nucleic acids can be analyzed by p~ùcedul~,s such as PCR, RFLP, AFLP, RAPID, norLl~n blots, Southern blots, dot blots etc. Proteins can be ~al~d by ELISA and western tt~chnitlu~c or by enzyme assays.
In one aspect, the system of the present invention can include a hollow tube having a .l~rl .. ~1 ,le region thereof adapted to receive a hic'~- 7~l satnple therein. An c.llbod~ule~t of such ahollowtubecanbestbed~ctrihedwithlGr~.~ucetotheFigures. Figures lA-lDshowthesystem 10 as inrlllt1ing a hollow tube 12 having a tubular or cyLindrical design which can be made of any water iLupe viuus deft rmQhl~ mQtt~ri~l Por ~YQnnrle~ in one emboAiment the hollow tube 12 is a plastic straw having accordion-like pleats 14 in the middle region of the hoLlow tube that permits bending or fle,Ying of the hollow tube. Suitable straws include NOISY BENDER brand straws and GLAD brand fle,Yible straws which are cul~ iaLly available. Alt~ a~ ly, other ~l~fnrmQhl~
hollow tube mQtl-riQlc which will find use in the present system include hoLlow ~iylhldcl, made of pc,l,- Lyl~e, pol.~ u~yle.lc, pol.~-,t~,l " pol.~ t~,harluOluc;Lylene, nylon, and the liLie. Preferably, the hoLlow tube h_s a fiexiblejoint or is bendable without forming a lsink in said hollow tube, or is bent wherein the bend causes a deviation from the k~n~tll~linQl axis e~t~n~ling through the center of said hollow tube is ~)n,f~,.dbly about 90~, but can range from 0~ to 180~.
As further shown in Figures lA-lD, the hollow tube 12 is configured to receive a tissue sample 15 cc ...p. ;~ filter paper 16 as a l~ tr for the tissue sample 17 e.g., leaf tissue. rne tissue sample 17 (a plant leaf) can be placed directly on the filter paper 16 and the paper folded to paCc the sample. In a pl~,f~lcd embo~lim~nt. the filter paper can include a dotted line 19 to direct a USOE on rl D~m~-nt of the fold. The amount of sample 17 placed in the filter papOE l~ L~k will normally be ~ e~ upon a number of factors wel~ ~o~.;~ d by those of ordinary skill in the art, ;~ l;..g the arnount of the ,~ e of interest present in, or the ~ ed extraction effi~irnl~y from, the sample. Where it is desired to extract nucleic acids from plant leaf c~m~ s, about 50-200mg of sample is usually ~ 7 ~Itho7l~h the hollow tube 12 can be cwL,LIu.;~ to much larger sample sizes. Once the sarnple 14 is pl.,l.~.,d and placed into the filter paper, it can be inserted into the hollow tube 12, preferably with no tissue satnple prottuding out of the p. . .,~;. "~1 end 13 of the hollow tube 12. Optionally, an eluant buffer can be iul~udu~d into the W O 97/18293 PCTrUS96/17951 hollow tube 12 to f~rilitilt~ efficient recovery and/or the stability of the ~ hS~ ce of interest. For , the filter paper can be soaked with an eluant buffer prior to insertion into the hoaaow tube assembly. ThefilterpaperthencanservetoproYidea~ vo~ of eluantin;.. e~ t~.contactwith the sample.
~ S The hollow tube 12 is preferably constructed of a material which is i~, ~le to the re of interest. This serves to protect the .~ of the system from being ~ t~
with the ~ a~;~ed ~ b~ , and to prevent the ~ ~ce from being lost prior to crmpl~~ing the CU~ IG'7SiOI1 phase of the method. It foaaows that if an eluant buffer is used, that the hollow tube 12 should preferably be; ~ hle to the buffer as well as the ~,. .hs" i,. ..~e of interest. In a preferred ~ h~l;. . .~. .l, the tissue sample is placed on a filter paper l6 prior to insertion of the sample into the hollow tube 12. The filter paper 16 can fi~rilit~ loading of the sample into the hollow tube, or can prevent the tissue sample from being sqll~oç7~d out of the hollow tube 12 during c.~ r~sion of the tube and sample using the cu...~ssion means. As A~sr.rihcd herein, the cvl~ lr ,sion of the hollow tube and sample is referred to as the ''eYtr~ctir~n step".
Thepresent hollow tube must have at least one ~ler~.... ~fblr region wherein the sample can be ~licrosçtl The rest of the hollow tube may be flexible or rigid, provided that it does not interfere with the ability of the ~,Ull~lG~7.7illg means to Culll~lrSS the ~leforrnilhle region of the hollow tube and express the 5~.1 ,cI,~.-r~ of interest from a samp}e lis~,os~d within the hollow tube.
Another embodiment of the subject invention c- ~- ~r~- . .C a hollow tube assembly having a liner disposed therein or integral LLv~rvwilll for retaining debris from a sample, whilc p~ 1;. .g the liquid and ~ of interest ~ ~ilecl from the sample to pass lI.e.dLu~l.. The liner, which can be filter milter~ inr~nrlin~ paper, cloth mesh, or polymer, can serve a similar purpose to the filter paper in which a sample, e.g., a leaf, is placed, as desrrihed hcl~in~uvr. An advantage of the hollow tube a.-,s~l~ly having a liner di ,~JoS~ therein is that the sample can be simply placed in the hollow tube without prior ~ ,~aLion of a filter paper. The hollow tube assembly having a liner Aicpose~l therein is preferably a .lf~r~" ~--~I le material as ~l~srribed herein for other ~mhorlimrntc of the hollow tube. In a preferred lomho~im~nt the hollow tube a;,~ bly can be ~ y a flattened conical shape, forming a funnel-shaped recept~rl~ for receiving the biol~gic ~I sample, and having a liner which .,..1.c~ lly covers the inner wall of the tube assembly and is closed at its vertex to prevent retained sample debris from mixing with the liquid or ~ b~"; ~~c of interest t~t~ ;kid from the sample. The conical hollow tube assembly can have open or closed ends, but p~,f~ly has at least one open end, and more lJ~ ~ly, has a top end (the wider end of the cone) that is open for ,~;~ iug the sample. In a most ~,~f~,.. ~l e",l~o.l;.. l the hollow tube assembly has a top end and a bottom end, both of which are open.
W O 97/18293 PCTrUS96/17951 ln one I ~ 1, the conical hollow tube assembly can be formed by sealing together two or more ~.lb,,~ y tri~n~nlz~r pieces of a liquid~ hle material to form a conical hollo~v tube .w,,~ul~ly. The pieces of the conical hollow tube assembly can be a&ered by any known li~luid-impermeable adLe~iv~; or can be of a material that a&eres to itself when heated, such as melting together at the points where heat is applied, thereby foIming a heat-seal. In a further f?mhor~im~nt where the hollow tube is fonned by adhering together more than one piece of m~tPri~1 at least one of said pieces of material can be longer than the other(s) such that the top end extends beyond the top end of the other pieces, forming a flap or tag. This tag can have a label or similar irlPntifi~r placed or st~mpe~7 thereon to serve as a marker or jrl.ontifiP.r of the sample source. Preferably, the tag is rlrl s-~ ble from the hollow tube assembly, e.g., by pti, rc" a~iOnS allowing the tag to be easily r1r~ r-d along the perforation line. The label or i(lentifirr affixed thereto can be m~trhr~d to a second, cu,~ o~ ,g label affixed to the body of the conical hollow tube assembly. In a most ~l~,f~,~l ~ ' ~ the conical hollow tube assembly and the .1~ le tag have m~trhin~ bar-code Labels for idt;l-tirying the sample source and sample. For ex~mr1c in ~la~ ;~g the illv~uliun using this Pmh~lim~nt ofthe subject a~aldflL" a sample collector obtaining a ".1~ r, of interest from a plant can identify an appropriate plant from which the sample is to be taken, remove a leaf sam~le from the source plant, and place it in the conical hollow tube assembly, detach the p~rfr~r~tPIl tag and attach it to the source plant to mark or label that plant c -.~ . ..l with the sample taken th~ rlul~l. The conical hollow tube assembly embodiment is illu~Llaled in Figure 8.
Adv~nt~oll~ly, by using the embodiment of the conical hollow tube ass~llbly having a liner ~,~ûsed therein, the assembly can be stored and shipped ,..1,~ 1y flat, can be easily transported in the ~eld, can fi~ri1it~t~- col1r-rti~n of samples by allowing a collector to open the flat assembly and place a sample directly inside the hollow tube assembly, can fi~ri1jtr A labeling of sample source and sample, and can ~1;,..;-- ~e the need for separate filter paper in which samples are wrapped. Furlher, whereby this conical hollow tube t .. 1~or1;~,1f .~1 can f~rilit~t~ use in the field, a buffer packet can also be in~l11de~l Preferably, the desired embodiment can include a puw~ cd bufferc~.. y.. r--1thatcanbelt;c~ t;;~withwater~orcanbe~lo~liaLclrmixedwiththefluid from the sample such that added water for lrf~ the buffer is not needed.
The present systemfurtherc ~-.y~ cs a culll~r~,s~iug means which operates in ~ ;r)n with the present hollow tube to deform the region c-~ g the sarnple, thereby Co~ g the sampleandc-pF1liilglll~ Gulnatleastaportionofthe~ e.ofinterest. One~nholim.-.ntof a Cu~ ,lllg means, e.g., a pair of rollers which can engage the hollow tube in a manner so as to COIll~lCS~ the hollow tube and a sample .li$p~ ~ed therein, is shown in Figure 2.
W O 97/18293 PCT~US96/17951 More speçifir~lly7 the present device 20 shown in Figure 2 can include a pair of rotatable rollers 21 and 22 j--xt~posffl so that they counter-rotate relative to each other. Each roller has a gear 23 and 24 respectively, attached at one end. The gears 23 and 24 mesh with each other, thereby f~rilit~ting the rollers' counter-rotation.
At least one of the gears 24 can be c .~ ;d directly or i.,~ ,tly to a drivirlg means (not shown) which drives the rollers 21 and 22. The driving means, which can be inr~ lF~ as part of the subject ~uv~uLioll~ can be adapted to be m-.tf.ri7Pd and, for eY~mple, to run at various speeds and in different directions, as the hollow tube 12 translates between rollers 21 and 22. Alternatively, the d~iving means can be a hand-crank or battery-driven ,..P~ ,,, useful in field applir~tion~ where el~-~tririty may not be available.
In ~ ition it is considered routine to provide a feature for aAju~ting the pressure exerted between the rollers, and to control such pressure n~ ;c~lly to, for PY~mplP, ~ ;I.IA;~ constant pressure as the portion of the hollow tube having sample ~ posed therein passes between the rollers 21 and 22.
Rollers 21 and 22 are preferably made of a rigid material such as hard plastic or metal.
II~,wc;~,.,., in certain aprlir~tir~nc~ rollers of a f~n, slightly d~r~.. ."~1,,1e material such as hard rubber may be more effective as would be ~pcu~.lL to one skilled in the art in view of the ~ h i ~ herein.
In the normal operation of device 20, rollers 21 and 22 initially contact each other. As the hollow tube 12 L. ~la~s between the rollers, they remain in ~ l -s~ A~ l contact, except in the region where theirc~ h.-l;.. grP~ n~hirisi~ lluyledbythetr~n~1~tinghollowtube12passingtht;.~b~
In an alt~nate ~'..b~~ -1, rollers 21 and 22 can be p~iti~np~l to have a gap between them, and the system will ~ L.~ly be adapted to allow the operator to adjust the gap size. An ~ u~lial~; gap .l;...~..~;..l. is selected dFpPnAing on the pa~ lal sample and hollow tube m~teri~l As the sample-cr~nt~ining area 26 of the hollow tube 12 passes between the rollers 21 and 2~ 22, the tissue sample is cu.u~ ssed arld fluid ~ the b~ e of interest flows bac~ ls toward the digal end 25 of the hollow tube 12. The fluid can be ~llP~ctpd in a cQllPction means 28 that can optionally contain a glass a~ pape~ filter element 29. Various other column packing ... ,. I ~ c can be used as well dq F.~l;..g on the ~b~ e of interest. The bottom end 27 of the cnllP,r,ti~n mea~s 28 can be adapted to F ~ the collPCtPd iluid. For ~ , the bottom end can be open 3û or can be stopped or fitted with a valve in order to allow an operator to discard the fluid as desired.
If the s~b:,l; ~~re of interest remains on the filter element 29 then the fluid is discarded. If the e of interest remains s--hlbili7~ in the fluid, then the fluid can be cnllPcted or retained for further ~ -n or analysis.
W O 97/18293 PCT~US96/17951 Figure 3A shows a cnlk~tinn means or coll~tinn tube 30 that has a top end 31, that is open to t~ceive the fluid from the hollow tube after the sample is c..~ s~;d, and a tapered, bottom end 32 tbat can be closed. In a preferred t .. 1~o.l;l.. 1 the closed bottom end can be scored to f~rilitPfe breaking off the closed end at a desired time to form an open bottom end of the cnl1t ctic~n tube.
S Optionally, a filter element 33 can be pocitinn~d toward the tapered end. The filter element 33 can be filter paper or glass fibers or the lil;e c~ p~ d into the tapered end 32 or preferably held into place by an O-ring 34. Alt~lldti~Gly, as il~l~ctr~P~ted in FIG. 3B the filter element 33 can be held into place by porous polyethylene discs 34 and 36. The plant source of an individual sample, the hollow tube (straw) and the cnll~ tube can be labelled, e.g., bar-coded, for audit and research ~w~oses.
Such lPh~lling can make it easy to identify plants or can fiP~ri1it1tt? ~.,tt~ p, the sample or source plant with the analytical results.
Figure 4, a ph~lu~a~h, shows a preferred embodiment of the present invention in a plucedwc; to isolate plant nucleic acids from leaf tissue. One end 40 of a flexible straw is loaded withaleafsamplec....l-P.;.~linafoldedfilterpaperthatwaswettedwithanuc}eicacid~-,tr~P~rtion solution. The loaded end of the straw 40 is run thlough the C~lllpl'~,~illg means, namely, the rollers 42 and 43, which are driven by an electric motor (not shown). Intracellular plant fluid is ("~rcsscd or ~ ,1 from the leaf sample and exit the hollow tube at the distal end 44 into a col~ tion cube tube 45 that contains a Whatman glass fiber filter 46 held into place by porous polyethylene discs above and below it. The coL~ected fluid can opti~n~lly be ;~ baled at a tt;llly~ldLwe elevated above or lowered below room ~la~ , which is typically arc~pttod by the ol~ lal;ly skilled artisar~ to be about 25~C. The nucleic acids in the e.~ ,s:,~,d fluid are then pl~ .;n l~,d by adding an alcohol, e.g., ethanol, or an adjacent hnnn~loglle or poCifinn~l isomer thereo~ The fluid is then mixed and allowed to stand for about 10 minutes. The fluid is disc~~~d by b 1 g offthe scored tip of the tapered end of the colloction cube tube or poking a hole in the tapered end. Fluid is forced t-h-rough the filter elernalt by ~l .1.; r. .,f I force, the applir~tion of pressure to the open end, or the applir~~inn of vacuum to the ~ w~,d or ~ lwise opened erld. The filter element is then washed with an alcohol wash solution. The pl~ Ird nucleic acids are then redissolved with a wallll aqueous bufferandelutedfromthe filter. The b..l.~ .l;PIIypure nucleic acids are then analyzed c,~lc~yillg standard terhni~les FIG. ~, a photograph, shows a 96 well c-.. l; ;.. ~- or mega titre tray 51 that contains two cnll~inn cube tubes ~i2 and 53. The cube tubes S2 and 53 fit snugly into the tray 50 and the whole unit can be spun in a c~-nfrifilge to spin o~f liquid fiom the bottoms of the cube tubes ~;2 and 53.
FIG 6, a 1.1.. .~-~ a~ , shows an empty cnllectic.n cube tube 61 with an open top end 62 and a closed bottom end 63. A filter paper 64 having a L~.l~ ,. of à~ . Iy the ~l;~k~. of the inner wall of the cube tube towards its closed end 63 is sandwiched between two discs of porous polyethylene 6~ and 66. The ~ ;~ed filter element is then placed into the open end 62 of a cube tube 61 and pushed down to the closed end 63 as depicted by loaded cube tube 67 (left side of photo). When the loaded cube tube 67 receives a liquid sample from the hollow tube assembly in c~)n~ etin~ the present extraction process, the liquid remains above the s~dwi~,Led filter element.
The liquid will pass through the filter paper when the cube tube is c~ ~l ~; r ~r~ or when pressure or vacuum is applied. The bottom end of the cube tube can be opened, as ~os~nbe~l to allow the liquid to be c~llecte~ if desired, or discarded if not needed.
FIG. 7, a photograph, depicts an extraction kit (minus buffers) ~ ~ lJ~ g a hollow tube, e.g., flexible plastic straw 71, a filter paper 72 and a loaded ç~ çti~-n cube tube 67. A ruler is shown on the left side of the photo to give an idea of the size of typical kit cu..l~vllt.ll~. Bu~ers are added and will depend on the sarnple and the s~lbst~nre of interest desired to be .,~ ,t~l.
FIG 8 is a photograph of the conical hollow tube embodiment showing the ~ nl~llo label and ~,. . ~ 1in~ 1abel on the body of the hollow tube. The emhoclim~nt shown is open at both top and bottom ends.
The following; . 1~ serve to illust~ate certain preferred embo~in~-ntc and aspects of the present ulvr~liwl and are not to be cvl~LIued as limiting the scope thereof.
F.x ~ fll In the, -l~ .lu:iul~ which follows, all weights are given in grams (g), milligrarnc (mg), ll~elu~ (,ug) or moles (mol). All c- ~ .c~ n.c are given as percent by volume (%), molar (M), millimolnr (mM) or rni~,lulllola~ (~M). All volu_es are given in liters (L), milliliters (mL) or microliters (~L), unless O~l wi~e in~i~ .tf rl The following e ,~ es ~lrmonctrate the operation of the system of the present invention to e~clract DNA and RNA from a plant leaf sample and should not be co~LI ued as limiting its scope.
Fxamrle l: Solutions The following buffer sol~ltinn~ were employed:
Nucleic Acid F Y ~ ;v. ~ Buffer: l ûO mM Tris-HCl(pH 8.0), l 0 ~nM Ethy~ acdic acid (EDTA), 10 mM ethylene glycol-bis(BB ~--.. ;.. o~ll.yl)N,N,N-tetracetic acid, 10 mM l,10-ortho-pl,....,...~ ; lc, 1.5% polyvinylpyrolidone 40, 1% sodiu_ dodecyl sulfate and 10 ug/ml pL~le ~lasc K.
lO M n~ Oil;~ acetate 100% Isol..u~)a.lol W O 97/18293 PCT~US96/179SI
l~thanol wash: 70% ethanol, 1 M .~.. ~.";.. acetate RNAse/Ethanol wash: 70% ethanol, 1 ~ ;~ acetate, 0.2 ug/ml DNAse-free RNAse A
TE Stora~ee Buffer: l0mM Tris-HCl (pH 8 0), 0. l rnM EDTA
S Fx~mple 2: Plant DNA E~LIa4Li~ln A hollow tube was ~1~1 by cutting the long end of a flexible plastic straw (GLADbrand) about 2.5 inches from the accordion-like pleats. A maize leaf sample of about l-l/2 crn x l0cm was folded and placed on a 3mm Whatrnan filter paper. The filter paper was folded l~ ll.wi~c with the leaf tissue on the inside. The folded paper was then inserted into the end of the hollow tube being careful that no leaf tissue was protruding out of the end of the hollow tube.
DNA ~, -1. <.. J ;. .1 ~ buffer (500 IlL) was added to the hollow tube near the end where the plant tissue sample was located thereby wetting the filter paper. The hollow tube is held hfJli~lllal when adding the DNA ~ buffer to keep the buffer in close ~/u~il~Ly to the plant tissue. This end was then colll~ ed by passing it through a roller assembly as shown in Figures 2 and 4.
The c~ ed sample was co11~ctf~-d in the specially adapted tube and ;nf ~lb~tf~A for 30 minuteS to allow Cf mp1ete protein rli~f-.ction and nucleic acid 501uhi~ on The sample can be stored- ' ~ lyinthisformifkeptinthedark. Thesamplewasadjustedto2.5 mM~.~.. ;.~
acetate by the addition of one third volume of l0 mM ~ ;.. acetate and then p~ .lrd with the addition of an equal volume of i~u~lu~l. The tube was ~uu.,LulGd and the ~ cl nucleic acids were captured on the Whatman GFB filter paper as the solution was cf-ntrifi1~d through the filter. The filter was washed 3 times with an ethanol wash (flf sf rihed in F~c~mp!e l). Total nucleic acids (i.e., DNA and RNA) were eluted with the filter with hot (6~ ~C) TE storage buffer. RNA free of DNA or, collvcl~cly DNA free of RNA, can be ~ d from the total nucleic acid sample by means well known to one of ordinary skill in the art. One means to make RNA-free DNAse is to wash the filter with an RNAse/Ethanol wash prior to elution of the DNA.
In order to verify that the extraction method was ~ r..l the DNA ~ ,;, are dissolved in a DNA storage buffer (lOrnM Tris-HCl, pH 8.0, l0mM NaCl, lmM EDTA3.The ~ of DNA orRNA ~ l may be f~ rd by methûds well known in the art. The quality of the c;,.L.~,L~d DNA can be ~~ .f-~l by size (e.g., >~0,OOObp) and by its ~iLiviLy to various ~ .......... f ~ If ~f~.. Ir:lCf-C under standard ef)nfliti~ n~ The quality of RNA may be f1~ cl by absence of f~F~ .~ l ;f n and other means well known in the art.The des~,lipLion of the preferred fmboflim~-ntc is not intf~nfled to limit the scope of the claimed invention. It will be evident from the following claims that the claimed invention irlcludes mf~ifif~tif n~ which would be obvious to a person of ordinary skill in the art. One can readily see W O 97/18293 PCTrUS96/179~1 that the present UlVt~llLi~1~ avoids a lot of transfer steps and c~ ",~1l ;nn of hald~ale. The present il~v~liull is basically a one tube process that serves as a field coll~ctinn tube and ~ if icdlion system - that dec.. ,ases c~ A1 ;nn and reduces the chance for mi~l~hf~11in3~ errors.
Cross-Reference to a Related Appli~tinn This is a c ,~ nl;on-in-part of co-pending application Serial No. 60/006,626, filed ~ 5 November 10, 1995.
Field of the Invention The present invention relates to the PYfrActinn of ~ ~ped s~ ln.~rcs from the Sulluulldill$ material in a hiol<~AI satnple and, more particularly, to a system and method fûr 0 CUlll~ illg a hinlclgicAI sample to extract a b~bbin-.~e of interest. ~dtlitinnAlly, the present invention relates to an extraction kit used to practice the present extraction process.
Back~round of the Invention Separation and extraction l~ ~ .es to obtain ~hPmi~AI or binlc.gi~-AI bul,s~lces from a kinl<gicAI sample are commnn p-w~s to many research, ~liA~n,cti~" IL~la~Jc~lLic and cc~ ~ah ~., 'v~s. Fl~i4u~lllly, a ~ e of interest will need to be l~llluv~d from a material matrix in order to fiA~ilit~ se.l~;nn for analysis. For P~Amrl~, plant biology .t;se~u~llers often desire to extract x~ ;... Pc, such as nucleic acids and proteins, found in plant leaves or other tissues. Such sllhstancff~ct extraction of the s~llLJsi n.-.~e The s--hsfAnre may also be physically c~.~.ay~ed by the solid matrix ~e.g. cell walls~, making even lengthy elution periods inA-le~nAte to extract the b~hb~ e from the matrix.
~otnri7Pcl roller devices have long been used to express b'.b.~ 'C such as cell sap from plant tissues. IIv.._~,., this apl!.oacl- is not well-suited when many, small samples need to be al~,l because it is slow and messy and the rol}ers must be cleaned and, in certain ;..x~ c ~ rd between sample runs. Also, it is difficult to collect the expelled ~l,bl A~ ~rc and particularly so to obtain ~ r; Vl;i extraction.
In the extraction of nucleic acids (DNA, RNA) from plant tissues, cnmrlicAtP~ and time c .. x.. ;.. g methods hinder an efficient extraction and purifirAtinn process. For P~ Ample, plant tissues are norrnally physically hnmngPni7p~d in a buffer which sol--hili7Pc the nucleic acid.
~mogP.ni7Atinn is a~mplixhpd by such means as a mortar and pestle, a food grinder or a hnmogPni~r (POLYTRON brand from n~ . ~PrhAnic~Al disruption is often ~1iffirult with very small ~nAntitiPC of plant tissue making it difficult to work with young plants.
After grinding, nucleic acids are then ~ led from the tissue slurry in a process that may involve multiple organic solvent P~innC ~chloroform, phenol, etc.) and centrifi-~Atinnc This is not only CA 02237329 1998-05-ll W O 97/18293 PCT~US96/17951 tcdious work but is also time c~ .;.-g Cross cn~ ;nn is a major concern with ml~r,h~mir,~l grinding as analytical f~chniq~ , e.g. PCR, become more ~clL7ilive Bnef Summary of the Invention The ~;ALIa~;Liull system ofthe present invention allows the efficient extraction of a snhst~nre of interest from a bil logir~l sample in minimal time, and with a minimal amount of eluant buffer.
Mess and system c ~ nn are ~ -;~ P~l by c-~ ;..;..g the sample and eluant buffer in a hollow tubular assembly, such as a plastic straw, such that tl~e sample never comes in contact with certain other c-....l.u.~ of the extraction system that are adv~nt~g.o~ lely kept from be~,....-;-.g c~ ln~
In ane aspect of the present invention the system includes a hollow tube assembly adapted to contain a sample and a means for cnllP,cfing the ~ e of interest. A Cc,lL~ g means operates in c-~. . .k; ~ r I ;nn with the hollow tube assembly to deform the region of the hollow tube assembly c.~ ;. .;. .g the sample, thereby cc)lllp~ ~g the sample and ~.Ypellin~ at least a portion of 1~ the ~ n. .. ~ of interest L~ L Ulll which is cc llecte~l Once collecte~1 the ~ of interest may be further purified and analyzed employing standard ~ ica~ion and analylical terhniqll~e A further aspect of the present invention involves a method of using the e~trFIrtinn system to separate the ~,..l.s~ .cG of interest from the sample. The extraction method is initiated by plVV' " g a hollow tube assembly havirlg a flefr- -- ' ' ~ region forc- ....n;~ the sample. The sample is placed within the ~.r~ ble region of the hollow tube assembly. ColL~ i.,A,iull means are then used to deform the hollow tube and ~ the sarnple: ~ ~ ' therein. The ~" ~1 .,e, 1~ ~r c of interest is then s~ nl~1 from the iiquid expelled from the sample, purified, and analyzed ernploying known ~iLcalion and analytical te~lmiques Of particular interest in the practice of the present illv~ulioll, an extraction kit is ~r~al~l and sold to those i ll~lc.,led in ~AIla iLillg ."~ 3 ~rP s of interest (DNA, RNA, proteins, etc.) from bifl~L, ' sarnples such as plants. The kit c. ~ ; all of the m~tf .ri5l1c, with the RY~f~ptinn of the co.ll~les,;on means, to enable one to carry out an efficient ~"~ha_liVIl process acculding to the present .. ~ iulL The kit will typically include a hûllow tube assembly (straw), a filter paper to hold the sample, a coll~,ti~n tube with packing and buffer solnti~ns to ~ta~ili7~?~ extract and/or purify the ~. .l .si ~ e of interest.
Rri~.f J~escription of the Drawings Figures lA-lD are a ~ f .~ n of the process of placing a bi~l~gic~l sample (plant leaf) Ul filter paper, and insertion thereof into a hollow tube a~ bly;
Figure 2 is a 1~ '71'~ ti~n of one embodiment of the system of the present invention;
Figures 3A and 3B show embodunents of the c~l1Rction means having filter discs disposed therein, FiglFire 4 is a photograph depicting the pPrfnrrn~nce of a method of the present invention.
~ 5 Figure S is a photograph of a 96 well pl~fnrm c~ .;.l;.. g two cn11ectinn tubes.
Figure 6 is a phntograrh showing an empty cnllPcti- n tube (right), a C~ llec.tir~n tube fully loaded with a filter disc (left), and the Cn~ U ~- ~~ ; of a filter, i.e., filter paper sandwiched in bctween two discs of porous polyethylene.
Figure 7 is a p1.~.1.~ ,.i.1~ of a hollow tube assembly, a filter paper to hold the tissue sample and a co11~cti. n tube.
Figure 8 is a photograph showing an Pmhor1imRnt of a conical hollow tube assembly according to the subject invention.
Detailed Disclosure of the Invention The present invention involves an a~pdLaLu~ and methods of use for co11ectin~ a ~.h~ e of interest c- ...1 i.;..~d in a biok~r~1 sample. The a~l~aLus of the subject invention c~....l.. ;~f c; a hollow tube assembly (h~ ~arL~l referred to as "hollow tube") for receiving a sample in a d~f()rm ~hle region thereof, a C~1~)1~i'7 7illg means for culn~n~ g said region having the sample thOEein, and a means for collPcting the ~ ~bs~ e of interest for b~ se~ -l p-lrifi~ti(m or physical, ~ ,- 1, or chPm~ l analysis. The cu.ll~l~s~ion of the sample by the Cu~ iug means bursts or disrupts the sample matrix (e.g., cells) thereby releasing the ~..l.~l,...- e of intOEest (e.g., cell c~.-l. .a.;) from the matrix into a buffer ~lRci~Pd to stabilize the specific b~l)71i~ac. of interest.
C~ ,;~. of the sample can furlhOE express the liquid c- .. ~ the ~ e of intOEest out of the hollow tube and into a cn11Prti~n means (l~ aL~I "c< 1lpctir n tube") for ~d-liticm~
of the "~ e of interest. The purified ~b~ e of interest can then be analyzed employing standard analytical tca]-~
The claimed invention includes the system, mt?i~ning the physical Cl~ "" ..1~; in operable n (;".1.,~ an c~a~,Liull kit employed to practice the present invention), and the method of using the system to extract, purify and analyze a ~ e of interest from a sample. While the ~.. 1.o l;.". .,1.; ~i~c~ . ;hed herein include a roller ~alaLus as a CU~ illg means for CC~ .. lg the sample, other equivalent ~- F~ ..c for dPfnrming the f4 rc - ...ilhle region of the hollow tube, and thOEeby Culll~ hlg the sample c~ Pd therein, are c~ tR I such as, for P~rnmple7 a press, or other device known to the ordinarily skilled artisan. Likewise, fimrtinnil11y e~uival~,.lL
hald~e can be g~h~ d for the hollow tube a.,~mbly and sample cnllRr,tinn tube.
CA 02237329 1998-05-ll W O 97/18293 PCT~US96/17951 The present invention will be found usefi~l in many sitllAti~nc whe}e it is desired to extract a substQn~ e. of inte}est from a bielcgl.~Al material matrix in order to fAeilitAf~ suhseq~l.nt manipulation for analysis. For t.~Amrle, 7~ es such as nucleic acids, proteins, and other I) o~ P~ c found in plant tissues (e~g~ leaves) can be ~ cc~scrlllly s~ t~d from the plant tissues by ~u~lvy--lg the illvc-l~tion~ In a~dition, nucleic acids, proteins, viruses, bacteria, carboLy~aL~;s and hi.~ it'.AIc found in plant and animal tissues or other bi.~l. gicQI mQtrirçc can be s~ d and ~ -1. a~;L~I by rrQrtit~ing the present ~~lvellLioll. Nucleic acids can be analyzed by p~ùcedul~,s such as PCR, RFLP, AFLP, RAPID, norLl~n blots, Southern blots, dot blots etc. Proteins can be ~al~d by ELISA and western tt~chnitlu~c or by enzyme assays.
In one aspect, the system of the present invention can include a hollow tube having a .l~rl .. ~1 ,le region thereof adapted to receive a hic'~- 7~l satnple therein. An c.llbod~ule~t of such ahollowtubecanbestbed~ctrihedwithlGr~.~ucetotheFigures. Figures lA-lDshowthesystem 10 as inrlllt1ing a hollow tube 12 having a tubular or cyLindrical design which can be made of any water iLupe viuus deft rmQhl~ mQtt~ri~l Por ~YQnnrle~ in one emboAiment the hollow tube 12 is a plastic straw having accordion-like pleats 14 in the middle region of the hoLlow tube that permits bending or fle,Ying of the hollow tube. Suitable straws include NOISY BENDER brand straws and GLAD brand fle,Yible straws which are cul~ iaLly available. Alt~ a~ ly, other ~l~fnrmQhl~
hollow tube mQtl-riQlc which will find use in the present system include hoLlow ~iylhldcl, made of pc,l,- Lyl~e, pol.~ u~yle.lc, pol.~-,t~,l " pol.~ t~,harluOluc;Lylene, nylon, and the liLie. Preferably, the hoLlow tube h_s a fiexiblejoint or is bendable without forming a lsink in said hollow tube, or is bent wherein the bend causes a deviation from the k~n~tll~linQl axis e~t~n~ling through the center of said hollow tube is ~)n,f~,.dbly about 90~, but can range from 0~ to 180~.
As further shown in Figures lA-lD, the hollow tube 12 is configured to receive a tissue sample 15 cc ...p. ;~ filter paper 16 as a l~ tr for the tissue sample 17 e.g., leaf tissue. rne tissue sample 17 (a plant leaf) can be placed directly on the filter paper 16 and the paper folded to paCc the sample. In a pl~,f~lcd embo~lim~nt. the filter paper can include a dotted line 19 to direct a USOE on rl D~m~-nt of the fold. The amount of sample 17 placed in the filter papOE l~ L~k will normally be ~ e~ upon a number of factors wel~ ~o~.;~ d by those of ordinary skill in the art, ;~ l;..g the arnount of the ,~ e of interest present in, or the ~ ed extraction effi~irnl~y from, the sample. Where it is desired to extract nucleic acids from plant leaf c~m~ s, about 50-200mg of sample is usually ~ 7 ~Itho7l~h the hollow tube 12 can be cwL,LIu.;~ to much larger sample sizes. Once the sarnple 14 is pl.,l.~.,d and placed into the filter paper, it can be inserted into the hollow tube 12, preferably with no tissue satnple prottuding out of the p. . .,~;. "~1 end 13 of the hollow tube 12. Optionally, an eluant buffer can be iul~udu~d into the W O 97/18293 PCTrUS96/17951 hollow tube 12 to f~rilitilt~ efficient recovery and/or the stability of the ~ hS~ ce of interest. For , the filter paper can be soaked with an eluant buffer prior to insertion into the hoaaow tube assembly. ThefilterpaperthencanservetoproYidea~ vo~ of eluantin;.. e~ t~.contactwith the sample.
~ S The hollow tube 12 is preferably constructed of a material which is i~, ~le to the re of interest. This serves to protect the .~ of the system from being ~ t~
with the ~ a~;~ed ~ b~ , and to prevent the ~ ~ce from being lost prior to crmpl~~ing the CU~ IG'7SiOI1 phase of the method. It foaaows that if an eluant buffer is used, that the hollow tube 12 should preferably be; ~ hle to the buffer as well as the ~,. .hs" i,. ..~e of interest. In a preferred ~ h~l;. . .~. .l, the tissue sample is placed on a filter paper l6 prior to insertion of the sample into the hollow tube 12. The filter paper 16 can fi~rilit~ loading of the sample into the hollow tube, or can prevent the tissue sample from being sqll~oç7~d out of the hollow tube 12 during c.~ r~sion of the tube and sample using the cu...~ssion means. As A~sr.rihcd herein, the cvl~ lr ,sion of the hollow tube and sample is referred to as the ''eYtr~ctir~n step".
Thepresent hollow tube must have at least one ~ler~.... ~fblr region wherein the sample can be ~licrosçtl The rest of the hollow tube may be flexible or rigid, provided that it does not interfere with the ability of the ~,Ull~lG~7.7illg means to Culll~lrSS the ~leforrnilhle region of the hollow tube and express the 5~.1 ,cI,~.-r~ of interest from a samp}e lis~,os~d within the hollow tube.
Another embodiment of the subject invention c- ~- ~r~- . .C a hollow tube assembly having a liner disposed therein or integral LLv~rvwilll for retaining debris from a sample, whilc p~ 1;. .g the liquid and ~ of interest ~ ~ilecl from the sample to pass lI.e.dLu~l.. The liner, which can be filter milter~ inr~nrlin~ paper, cloth mesh, or polymer, can serve a similar purpose to the filter paper in which a sample, e.g., a leaf, is placed, as desrrihed hcl~in~uvr. An advantage of the hollow tube a.-,s~l~ly having a liner di ,~JoS~ therein is that the sample can be simply placed in the hollow tube without prior ~ ,~aLion of a filter paper. The hollow tube assembly having a liner Aicpose~l therein is preferably a .lf~r~" ~--~I le material as ~l~srribed herein for other ~mhorlimrntc of the hollow tube. In a preferred lomho~im~nt the hollow tube a;,~ bly can be ~ y a flattened conical shape, forming a funnel-shaped recept~rl~ for receiving the biol~gic ~I sample, and having a liner which .,..1.c~ lly covers the inner wall of the tube assembly and is closed at its vertex to prevent retained sample debris from mixing with the liquid or ~ b~"; ~~c of interest t~t~ ;kid from the sample. The conical hollow tube assembly can have open or closed ends, but p~,f~ly has at least one open end, and more lJ~ ~ly, has a top end (the wider end of the cone) that is open for ,~;~ iug the sample. In a most ~,~f~,.. ~l e",l~o.l;.. l the hollow tube assembly has a top end and a bottom end, both of which are open.
W O 97/18293 PCTrUS96/17951 ln one I ~ 1, the conical hollow tube assembly can be formed by sealing together two or more ~.lb,,~ y tri~n~nlz~r pieces of a liquid~ hle material to form a conical hollo~v tube .w,,~ul~ly. The pieces of the conical hollow tube assembly can be a&ered by any known li~luid-impermeable adLe~iv~; or can be of a material that a&eres to itself when heated, such as melting together at the points where heat is applied, thereby foIming a heat-seal. In a further f?mhor~im~nt where the hollow tube is fonned by adhering together more than one piece of m~tPri~1 at least one of said pieces of material can be longer than the other(s) such that the top end extends beyond the top end of the other pieces, forming a flap or tag. This tag can have a label or similar irlPntifi~r placed or st~mpe~7 thereon to serve as a marker or jrl.ontifiP.r of the sample source. Preferably, the tag is rlrl s-~ ble from the hollow tube assembly, e.g., by pti, rc" a~iOnS allowing the tag to be easily r1r~ r-d along the perforation line. The label or i(lentifirr affixed thereto can be m~trhr~d to a second, cu,~ o~ ,g label affixed to the body of the conical hollow tube assembly. In a most ~l~,f~,~l ~ ' ~ the conical hollow tube assembly and the .1~ le tag have m~trhin~ bar-code Labels for idt;l-tirying the sample source and sample. For ex~mr1c in ~la~ ;~g the illv~uliun using this Pmh~lim~nt ofthe subject a~aldflL" a sample collector obtaining a ".1~ r, of interest from a plant can identify an appropriate plant from which the sample is to be taken, remove a leaf sam~le from the source plant, and place it in the conical hollow tube assembly, detach the p~rfr~r~tPIl tag and attach it to the source plant to mark or label that plant c -.~ . ..l with the sample taken th~ rlul~l. The conical hollow tube assembly embodiment is illu~Llaled in Figure 8.
Adv~nt~oll~ly, by using the embodiment of the conical hollow tube ass~llbly having a liner ~,~ûsed therein, the assembly can be stored and shipped ,..1,~ 1y flat, can be easily transported in the ~eld, can fi~ri1it~t~- col1r-rti~n of samples by allowing a collector to open the flat assembly and place a sample directly inside the hollow tube assembly, can fi~ri1jtr A labeling of sample source and sample, and can ~1;,..;-- ~e the need for separate filter paper in which samples are wrapped. Furlher, whereby this conical hollow tube t .. 1~or1;~,1f .~1 can f~rilit~t~ use in the field, a buffer packet can also be in~l11de~l Preferably, the desired embodiment can include a puw~ cd bufferc~.. y.. r--1thatcanbelt;c~ t;;~withwater~orcanbe~lo~liaLclrmixedwiththefluid from the sample such that added water for lrf~ the buffer is not needed.
The present systemfurtherc ~-.y~ cs a culll~r~,s~iug means which operates in ~ ;r)n with the present hollow tube to deform the region c-~ g the sarnple, thereby Co~ g the sampleandc-pF1liilglll~ Gulnatleastaportionofthe~ e.ofinterest. One~nholim.-.ntof a Cu~ ,lllg means, e.g., a pair of rollers which can engage the hollow tube in a manner so as to COIll~lCS~ the hollow tube and a sample .li$p~ ~ed therein, is shown in Figure 2.
W O 97/18293 PCT~US96/17951 More speçifir~lly7 the present device 20 shown in Figure 2 can include a pair of rotatable rollers 21 and 22 j--xt~posffl so that they counter-rotate relative to each other. Each roller has a gear 23 and 24 respectively, attached at one end. The gears 23 and 24 mesh with each other, thereby f~rilit~ting the rollers' counter-rotation.
At least one of the gears 24 can be c .~ ;d directly or i.,~ ,tly to a drivirlg means (not shown) which drives the rollers 21 and 22. The driving means, which can be inr~ lF~ as part of the subject ~uv~uLioll~ can be adapted to be m-.tf.ri7Pd and, for eY~mple, to run at various speeds and in different directions, as the hollow tube 12 translates between rollers 21 and 22. Alternatively, the d~iving means can be a hand-crank or battery-driven ,..P~ ,,, useful in field applir~tion~ where el~-~tririty may not be available.
In ~ ition it is considered routine to provide a feature for aAju~ting the pressure exerted between the rollers, and to control such pressure n~ ;c~lly to, for PY~mplP, ~ ;I.IA;~ constant pressure as the portion of the hollow tube having sample ~ posed therein passes between the rollers 21 and 22.
Rollers 21 and 22 are preferably made of a rigid material such as hard plastic or metal.
II~,wc;~,.,., in certain aprlir~tir~nc~ rollers of a f~n, slightly d~r~.. ."~1,,1e material such as hard rubber may be more effective as would be ~pcu~.lL to one skilled in the art in view of the ~ h i ~ herein.
In the normal operation of device 20, rollers 21 and 22 initially contact each other. As the hollow tube 12 L. ~la~s between the rollers, they remain in ~ l -s~ A~ l contact, except in the region where theirc~ h.-l;.. grP~ n~hirisi~ lluyledbythetr~n~1~tinghollowtube12passingtht;.~b~
In an alt~nate ~'..b~~ -1, rollers 21 and 22 can be p~iti~np~l to have a gap between them, and the system will ~ L.~ly be adapted to allow the operator to adjust the gap size. An ~ u~lial~; gap .l;...~..~;..l. is selected dFpPnAing on the pa~ lal sample and hollow tube m~teri~l As the sample-cr~nt~ining area 26 of the hollow tube 12 passes between the rollers 21 and 2~ 22, the tissue sample is cu.u~ ssed arld fluid ~ the b~ e of interest flows bac~ ls toward the digal end 25 of the hollow tube 12. The fluid can be ~llP~ctpd in a cQllPction means 28 that can optionally contain a glass a~ pape~ filter element 29. Various other column packing ... ,. I ~ c can be used as well dq F.~l;..g on the ~b~ e of interest. The bottom end 27 of the cnllP,r,ti~n mea~s 28 can be adapted to F ~ the collPCtPd iluid. For ~ , the bottom end can be open 3û or can be stopped or fitted with a valve in order to allow an operator to discard the fluid as desired.
If the s~b:,l; ~~re of interest remains on the filter element 29 then the fluid is discarded. If the e of interest remains s--hlbili7~ in the fluid, then the fluid can be cnllPcted or retained for further ~ -n or analysis.
W O 97/18293 PCT~US96/17951 Figure 3A shows a cnlk~tinn means or coll~tinn tube 30 that has a top end 31, that is open to t~ceive the fluid from the hollow tube after the sample is c..~ s~;d, and a tapered, bottom end 32 tbat can be closed. In a preferred t .. 1~o.l;l.. 1 the closed bottom end can be scored to f~rilitPfe breaking off the closed end at a desired time to form an open bottom end of the cnl1t ctic~n tube.
S Optionally, a filter element 33 can be pocitinn~d toward the tapered end. The filter element 33 can be filter paper or glass fibers or the lil;e c~ p~ d into the tapered end 32 or preferably held into place by an O-ring 34. Alt~lldti~Gly, as il~l~ctr~P~ted in FIG. 3B the filter element 33 can be held into place by porous polyethylene discs 34 and 36. The plant source of an individual sample, the hollow tube (straw) and the cnll~ tube can be labelled, e.g., bar-coded, for audit and research ~w~oses.
Such lPh~lling can make it easy to identify plants or can fiP~ri1it1tt? ~.,tt~ p, the sample or source plant with the analytical results.
Figure 4, a ph~lu~a~h, shows a preferred embodiment of the present invention in a plucedwc; to isolate plant nucleic acids from leaf tissue. One end 40 of a flexible straw is loaded withaleafsamplec....l-P.;.~linafoldedfilterpaperthatwaswettedwithanuc}eicacid~-,tr~P~rtion solution. The loaded end of the straw 40 is run thlough the C~lllpl'~,~illg means, namely, the rollers 42 and 43, which are driven by an electric motor (not shown). Intracellular plant fluid is ("~rcsscd or ~ ,1 from the leaf sample and exit the hollow tube at the distal end 44 into a col~ tion cube tube 45 that contains a Whatman glass fiber filter 46 held into place by porous polyethylene discs above and below it. The coL~ected fluid can opti~n~lly be ;~ baled at a tt;llly~ldLwe elevated above or lowered below room ~la~ , which is typically arc~pttod by the ol~ lal;ly skilled artisar~ to be about 25~C. The nucleic acids in the e.~ ,s:,~,d fluid are then pl~ .;n l~,d by adding an alcohol, e.g., ethanol, or an adjacent hnnn~loglle or poCifinn~l isomer thereo~ The fluid is then mixed and allowed to stand for about 10 minutes. The fluid is disc~~~d by b 1 g offthe scored tip of the tapered end of the colloction cube tube or poking a hole in the tapered end. Fluid is forced t-h-rough the filter elernalt by ~l .1.; r. .,f I force, the applir~tion of pressure to the open end, or the applir~~inn of vacuum to the ~ w~,d or ~ lwise opened erld. The filter element is then washed with an alcohol wash solution. The pl~ Ird nucleic acids are then redissolved with a wallll aqueous bufferandelutedfromthe filter. The b..l.~ .l;PIIypure nucleic acids are then analyzed c,~lc~yillg standard terhni~les FIG. ~, a photograph, shows a 96 well c-.. l; ;.. ~- or mega titre tray 51 that contains two cnll~inn cube tubes ~i2 and 53. The cube tubes S2 and 53 fit snugly into the tray 50 and the whole unit can be spun in a c~-nfrifilge to spin o~f liquid fiom the bottoms of the cube tubes ~;2 and 53.
FIG 6, a 1.1.. .~-~ a~ , shows an empty cnllectic.n cube tube 61 with an open top end 62 and a closed bottom end 63. A filter paper 64 having a L~.l~ ,. of à~ . Iy the ~l;~k~. of the inner wall of the cube tube towards its closed end 63 is sandwiched between two discs of porous polyethylene 6~ and 66. The ~ ;~ed filter element is then placed into the open end 62 of a cube tube 61 and pushed down to the closed end 63 as depicted by loaded cube tube 67 (left side of photo). When the loaded cube tube 67 receives a liquid sample from the hollow tube assembly in c~)n~ etin~ the present extraction process, the liquid remains above the s~dwi~,Led filter element.
The liquid will pass through the filter paper when the cube tube is c~ ~l ~; r ~r~ or when pressure or vacuum is applied. The bottom end of the cube tube can be opened, as ~os~nbe~l to allow the liquid to be c~llecte~ if desired, or discarded if not needed.
FIG. 7, a photograph, depicts an extraction kit (minus buffers) ~ ~ lJ~ g a hollow tube, e.g., flexible plastic straw 71, a filter paper 72 and a loaded ç~ çti~-n cube tube 67. A ruler is shown on the left side of the photo to give an idea of the size of typical kit cu..l~vllt.ll~. Bu~ers are added and will depend on the sarnple and the s~lbst~nre of interest desired to be .,~ ,t~l.
FIG 8 is a photograph of the conical hollow tube embodiment showing the ~ nl~llo label and ~,. . ~ 1in~ 1abel on the body of the hollow tube. The emhoclim~nt shown is open at both top and bottom ends.
The following; . 1~ serve to illust~ate certain preferred embo~in~-ntc and aspects of the present ulvr~liwl and are not to be cvl~LIued as limiting the scope thereof.
F.x ~ fll In the, -l~ .lu:iul~ which follows, all weights are given in grams (g), milligrarnc (mg), ll~elu~ (,ug) or moles (mol). All c- ~ .c~ n.c are given as percent by volume (%), molar (M), millimolnr (mM) or rni~,lulllola~ (~M). All volu_es are given in liters (L), milliliters (mL) or microliters (~L), unless O~l wi~e in~i~ .tf rl The following e ,~ es ~lrmonctrate the operation of the system of the present invention to e~clract DNA and RNA from a plant leaf sample and should not be co~LI ued as limiting its scope.
Fxamrle l: Solutions The following buffer sol~ltinn~ were employed:
Nucleic Acid F Y ~ ;v. ~ Buffer: l ûO mM Tris-HCl(pH 8.0), l 0 ~nM Ethy~ acdic acid (EDTA), 10 mM ethylene glycol-bis(BB ~--.. ;.. o~ll.yl)N,N,N-tetracetic acid, 10 mM l,10-ortho-pl,....,...~ ; lc, 1.5% polyvinylpyrolidone 40, 1% sodiu_ dodecyl sulfate and 10 ug/ml pL~le ~lasc K.
lO M n~ Oil;~ acetate 100% Isol..u~)a.lol W O 97/18293 PCT~US96/179SI
l~thanol wash: 70% ethanol, 1 M .~.. ~.";.. acetate RNAse/Ethanol wash: 70% ethanol, 1 ~ ;~ acetate, 0.2 ug/ml DNAse-free RNAse A
TE Stora~ee Buffer: l0mM Tris-HCl (pH 8 0), 0. l rnM EDTA
S Fx~mple 2: Plant DNA E~LIa4Li~ln A hollow tube was ~1~1 by cutting the long end of a flexible plastic straw (GLADbrand) about 2.5 inches from the accordion-like pleats. A maize leaf sample of about l-l/2 crn x l0cm was folded and placed on a 3mm Whatrnan filter paper. The filter paper was folded l~ ll.wi~c with the leaf tissue on the inside. The folded paper was then inserted into the end of the hollow tube being careful that no leaf tissue was protruding out of the end of the hollow tube.
DNA ~, -1. <.. J ;. .1 ~ buffer (500 IlL) was added to the hollow tube near the end where the plant tissue sample was located thereby wetting the filter paper. The hollow tube is held hfJli~lllal when adding the DNA ~ buffer to keep the buffer in close ~/u~il~Ly to the plant tissue. This end was then colll~ ed by passing it through a roller assembly as shown in Figures 2 and 4.
The c~ ed sample was co11~ctf~-d in the specially adapted tube and ;nf ~lb~tf~A for 30 minuteS to allow Cf mp1ete protein rli~f-.ction and nucleic acid 501uhi~ on The sample can be stored- ' ~ lyinthisformifkeptinthedark. Thesamplewasadjustedto2.5 mM~.~.. ;.~
acetate by the addition of one third volume of l0 mM ~ ;.. acetate and then p~ .lrd with the addition of an equal volume of i~u~lu~l. The tube was ~uu.,LulGd and the ~ cl nucleic acids were captured on the Whatman GFB filter paper as the solution was cf-ntrifi1~d through the filter. The filter was washed 3 times with an ethanol wash (flf sf rihed in F~c~mp!e l). Total nucleic acids (i.e., DNA and RNA) were eluted with the filter with hot (6~ ~C) TE storage buffer. RNA free of DNA or, collvcl~cly DNA free of RNA, can be ~ d from the total nucleic acid sample by means well known to one of ordinary skill in the art. One means to make RNA-free DNAse is to wash the filter with an RNAse/Ethanol wash prior to elution of the DNA.
In order to verify that the extraction method was ~ r..l the DNA ~ ,;, are dissolved in a DNA storage buffer (lOrnM Tris-HCl, pH 8.0, l0mM NaCl, lmM EDTA3.The ~ of DNA orRNA ~ l may be f~ rd by methûds well known in the art. The quality of the c;,.L.~,L~d DNA can be ~~ .f-~l by size (e.g., >~0,OOObp) and by its ~iLiviLy to various ~ .......... f ~ If ~f~.. Ir:lCf-C under standard ef)nfliti~ n~ The quality of RNA may be f1~ cl by absence of f~F~ .~ l ;f n and other means well known in the art.The des~,lipLion of the preferred fmboflim~-ntc is not intf~nfled to limit the scope of the claimed invention. It will be evident from the following claims that the claimed invention irlcludes mf~ifif~tif n~ which would be obvious to a person of ordinary skill in the art. One can readily see W O 97/18293 PCTrUS96/179~1 that the present UlVt~llLi~1~ avoids a lot of transfer steps and c~ ",~1l ;nn of hald~ale. The present il~v~liull is basically a one tube process that serves as a field coll~ctinn tube and ~ if icdlion system - that dec.. ,ases c~ A1 ;nn and reduces the chance for mi~l~hf~11in3~ errors.
Claims (24)
1. A system for extracting a substance from a biological sample, said system comprising (a) a hollow tube assembly having a deformable region; and (b) means for collection of the extracted substance.
2. The system, according to claim 1, wherein said hollow tube assembly comprises a liquid-impermeable material.
3. The system, according to claim 2, wherein said liquid-impermeable material is a polymer.
4. The system, according to claim 1, wherein said hollow tube assembly has a bendable joint capable of forming, or is bent to form, an angled hollow tube.
5. The system, according to claim 1, wherein said hollow tube is conical.
6. The system, according to claim 5, wherein said conical hollow tube has disposed therein a filter as a liner of an inner wall of said hollow tube.
7. The system, according to claim 6, wherein said filter liner is integral with the inner wall of said hollow tube.
8. The system, according to claim 1, wherein said collection means is a collection tube.
9. The system, according to claim 1, wherein said collection means further comprises a filter element disposed therein.
10. The system, according to claim 9, wherein said filter element is a filter disc.
11. The system, according to claim 9, wherein said filter element is held within said collection tube by an O-ring.
12. The system, according to claim 9, wherein said filter element is held within said collection tube by being sandwiched between two porous polyethylene discs.
13. The system, according to claim 1, wherein said system further comprises a compressing means.
14. The system, according to claim 13, wherein said compressing means comprises at least a pair of rollers which are engaged at sufficient pressure to compress the biological sample to express liquid therefrom, while allowing a hollow tube assembly to deformably pass therebetween.
15. The system, according to claim 13, wherein said compressing means is motor-driven.
16. A method of extracting a substance of interest from a biological sample, said method comprising the steps of:
a. placing a sample comprising the substance of interest in a deformable region of a hollow tube assembly;
b. deforming the hollow tube assembly in the deformable region that contains the sample whereby a liquid comprising the substance of interest is expressed from the sample; and c. collecting the expressed liquid.
a. placing a sample comprising the substance of interest in a deformable region of a hollow tube assembly;
b. deforming the hollow tube assembly in the deformable region that contains the sample whereby a liquid comprising the substance of interest is expressed from the sample; and c. collecting the expressed liquid.
17. The method, according to claim 16, wherein said substance of interest is selected from the group consisting of a nucleic acid or an amino acid.
18. The method, according to claim 17, wherein said nucleic acid is DNA or RNA.
19. The method, according to claim 1, wherein said biological sample is a plant tissue.
20. The method, according to claim 16, wherein said deforming step (b) employs a pair of engaging rollers.
21. The method, according to claim 16, wherein said method further comprises wrapping said biological sample in filter paper prior to placing the sample in said hollow tube assembly.
22. The method, according to claim 21, wherein said filter paper is soaked with an elution buffer.
23. An extraction kit which comprises:
a. a hollow tube assembly that is deformable at least at one end and which is adapted to receive a sample containing a substance of interest;
b. a means for collecting the substance of interest from said sample; and c. a buffer composition capable of stabilizing. extracting or purifying the substance of interest.
a. a hollow tube assembly that is deformable at least at one end and which is adapted to receive a sample containing a substance of interest;
b. a means for collecting the substance of interest from said sample; and c. a buffer composition capable of stabilizing. extracting or purifying the substance of interest.
24. The extraction kit, according to claim 23, further comprising a filter paper to hold the sample and wherein the collecting means (b) is a collection tube comprising an open end, a closed end, and a filter element or column packing.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US662695P | 1995-11-13 | 1995-11-13 | |
| US60/006,626 | 1995-11-13 | ||
| PCT/US1996/017951 WO1997018293A1 (en) | 1995-11-13 | 1996-11-08 | Extraction system and method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2237329A1 true CA2237329A1 (en) | 1997-05-22 |
Family
ID=21721811
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002237329A Abandoned CA2237329A1 (en) | 1995-11-13 | 1996-11-08 | Extraction system and method |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0866847A1 (en) |
| JP (1) | JP2000500651A (en) |
| AU (1) | AU7674296A (en) |
| CA (1) | CA2237329A1 (en) |
| WO (1) | WO1997018293A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109724844A (en) * | 2019-02-13 | 2019-05-07 | 江苏双双高新科技有限公司 | A kind of water drawing device of water environment sampling machine |
| CN109724845A (en) * | 2019-02-13 | 2019-05-07 | 江苏双双高新科技有限公司 | A kind of sampling robots sampler |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19916534A1 (en) * | 1999-04-13 | 2000-10-19 | Gsf Forschungszentrum Umwelt | RNA isolation kit |
| US20070116613A1 (en) * | 2005-11-23 | 2007-05-24 | Donat Elsener | Sample tube and system for storing and providing nucleic acid samples |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL187692C (en) * | 1982-07-06 | 1991-12-16 | Leonardus Anthonius Johannes M | METHOD AND APPARATUS FOR RECOVERING LIQUID FRACTIONS FROM SEPARATE SAMPLES FROM VEGETABLE OR ANIMAL MATERIALS, AND SAMPLE SUITABLE FOR THIS |
| DE59502334D1 (en) * | 1994-01-07 | 1998-07-02 | Qiagen Gmbh | METHOD FOR CRUSHING HIGH MOLECULAR STRUCTURES |
-
1996
- 1996-11-08 CA CA002237329A patent/CA2237329A1/en not_active Abandoned
- 1996-11-08 EP EP96939609A patent/EP0866847A1/en not_active Withdrawn
- 1996-11-08 AU AU76742/96A patent/AU7674296A/en not_active Abandoned
- 1996-11-08 JP JP9518951A patent/JP2000500651A/en not_active Ceased
- 1996-11-08 WO PCT/US1996/017951 patent/WO1997018293A1/en not_active Application Discontinuation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109724844A (en) * | 2019-02-13 | 2019-05-07 | 江苏双双高新科技有限公司 | A kind of water drawing device of water environment sampling machine |
| CN109724845A (en) * | 2019-02-13 | 2019-05-07 | 江苏双双高新科技有限公司 | A kind of sampling robots sampler |
| CN109724845B (en) * | 2019-02-13 | 2021-06-15 | 江苏双双高新科技有限公司 | Sampling device for sampling robot |
| CN109724844B (en) * | 2019-02-13 | 2021-06-29 | 江苏双双高新科技有限公司 | Water drawing device of water environment sampling machine |
Also Published As
| Publication number | Publication date |
|---|---|
| AU7674296A (en) | 1997-06-05 |
| JP2000500651A (en) | 2000-01-25 |
| WO1997018293A1 (en) | 1997-05-22 |
| EP0866847A1 (en) | 1998-09-30 |
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