EXTRACTION SYSTEM AND METHOD

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: EXTRACTION SYSTEM AND METHOD Cross-Reference to a Related Apphcation This is a continuation-in-part of co-pending application Senal No 60/006,626, filed November 10, 1995 Field of the Invention The present invention relates to the extraction of entrapped substances from the surrounding matenal in a biological sample and, more particularly, to a system and method for compressing a biological sample to extract a substance of interest Additionally, the present mvention relates to an extraction kit used to prac ce the present extraction process Background of the Invention Separation and extraction techniques to obtain chemical or biological substances from a biological sample are common prerequisites to many research, diagnostic, therapeutic and commercial objectives Frequently, a substance of interest will need to be removed from a matenal matnx in order to facilitate subsequent manipulation for analysis For example, plant biology researchers often desire to extract substances, such as nucleic acids and proteins, found m plant leaves or other tissues Such substancffect extraction of the substance The substance may also be physically entrapped by the solid matnx (e g cell walls), making even lengthy elution penods madequate to extract the substance from the matnx Motonzed roller devices have long been used to express substances such as cell sap from plant tissues However, this approach is not well-suited when many, small samples need to be prepared because it is slow and messy and the rollers must be cleaned and, in certain instances, sterilized between sample runs Also, it is difficult to collect the expelled substance and particularly so to obtain quantitative extraction In the extraction of nucleic acids (DNA, RNA) from plant tissues, complicated and time consuming methods hinder an efficient extracύon and punfication process For example, plant tissues are normally physically homogenized m a buffer which solubilizes the nucleic acid Homogemzation is accomplished by such means as a mortar and pestle, a food grinder or a homogenizer (POLYTRON brand from Brinkman Instruments) Mechanical disruption is often difficult with very small quantities of plant ssue making it difficult to work with young plants After gπnding, nucleic acids are then extracted from the tissue slurry in a process that may mvolve multiple organic solvent extractions (chloroform, phenol, etc ) and centnfugations This is not only tedious work but is also time consuming Cross contamination is a major concern with mechanical grinding as analytical techniques, e g PCR, become more sensitive Bπef Summary ofthe Invention The extraction system ofthe present invention allows the efficient extraction of a substance of mterest from a biological sample in minimal time, and with a minimal amount of eluant buffer Mess and system contamination are minimized by contaimng the sample and eluant buffer in a hollow tubular assembly, such as a plastic straw, such that the sample never comes in contact with certam other components of the extraction system that are advantageously kept from becoming contaminated In one aspect ofthe present mvention the system mcludes a hollow tube assembly adapted to contain a sample and a means for collecting the substance of mterest A compressmg means operates in combmation with the hollow tube assembly to deform the region of the hollow tube assembly contaimng the sample, thereby compressmg the sample and expellmg at least a portion of the substance of interest therefrom which is collected Once collected, the substance of mterest may be further punfied and analyzed employing standard punfication and analytical techniques A fiirther aspect of the present mvention involves a method of usmg the extraction system to separate the substance of mterest from the sample The extraction method is initiated by providing a hollow tube assembly having a deformable region for contammg the sample The sample is placed withm the deformable region of the hollow tube assembly Compression means are then used to deform the hollow tube and compress the sample contamed therein The substance of interest is then separated from the liquid expelled from the sample, punfied, and analyzed employing known punfication and analytical techniques Of particular mterest m the practice ofthe present mvention, an extraction kit is prepared and sold to those interested in extracting substances of mterest (DNA, RNA, proteins, etc ) from biological samples such as plants The kit compnses all ofthe mateπals, with the exception ofthe compression means, to enable one to carry out an efficient extraction process accordmg to the present inventioα The -at will typically include a hollow tube assembly (straw), a filter paper to hold the sample, a collection tube with packing and buffer solutions to stabilize, extract and/or pun y the substance of mterest Bnef Descnption ofthe Drawmgs Figures 1A-1D are a representation of the process of placmg a biological sample (plant leaf) in filter paper, and insertion thereof mto a hollow tube assembly; Figure 2 is a representation of one embodiment of the system of the present mvention, Figures 3A and 3B show embodiments ofthe collection means havmg filter discs disposed therein, Figure 4 is a photograph depictmg the performance of a method ofthe present mvention Figure 5 is a photograph of a 96 w ell platform contaimng two collection tubes Figure 6 is a photograph showing an empty collection tube (nght), a collection tube full) loaded with a filter disc (left), and the components of a filter, I e , filter paper sandwiched in between two discs of porous polyethylene Figure 7 is a photograph of a hollow tube assembly, a filter paper to hold the tissue sample and a collection tube Figure 8 is a photograph showing an embodiment of a conical hollow tube assembly accordmg to the subject mvention Detailed Disclosure of the Invention The present mvention involves an apparatus and methods of use for collecting a substance of mterest contamed m a biological sample The apparatus of the subject mvention compnses a hollow tube assembly (heremafter referred to as "hollow tube") for receivmg a sample m a deformable region thereof, a compressmg means for compressmg said region havmg the sample therein, and a means for collecting the substance of mterest for subsequent punfication or physical, biological, or chemical analysis The compression ofthe sample by the compressmg means bursts or disrupts the sample matnx (e g , cells) thereby releasmg the substance of mterest (e g , cell contents) from the matnx mto a buffer designed to stabilize the specific substance of mterest Compression ofthe sample can further express the liquid contammg the substance of mterest out of the hollow tube and mto a collection means (heremafter "collection tube") for additional punfication of the substance of mterest The punfied substance of mterest can then be analyzed employmg standard analytical techniques The claimed mvention mcludes the system, meaning the physical elements in operable combination (including an extraction -at employed to practice the present mvention), and the method of usmg the system to extract, punfy and analyze a substance of mterest from a sample While the embodiments descnbed herein mclude a roller apparatus as a compressmg means for compressmg the sample, other equivalent mechanisms for deforming the deformable region ofthe hollow tube, and thereby compressmg the sample contamed therein, are contemplated, such as, for example, a press, or other device known to the ordmanly skilled artisan Likewise, functionally equivalent hardware can be substituted for the hollow tube assembly and sample collection tube The present invention will be found useful in many situations where it is desired to extract a substance of mterest from a biological mateπal matnx m order to facilitate subsequent manipulation for analysis For example, substances such as nucleic acids, protems, and other biochemicals found in plant tissues (e , leaves) can be successfully separated from the plant tissues by employmg the mvention In addition, nucleic acids, protems, viruses, bacteπa, carbohydrates and biochemicals found in plant and animal tissues or other biological matπces, can be separated and extracted by practicing the present mvention Nucleic acids can be analyzed by procedures such as PCR, RFLP, AFLP, RAPID, northern blots, Southern blots, dot blots etc Proteins can be analyzed by ELISA and western techniques, or by enzyme assays In one aspect, the system of the present mvention can mclude a hollow tube havmg a deformable region thereof adapted to receive a biological sample therein An embodiment of such a hollow tube can best be descnbed with reference to the Figures Figures 1 A- 1 D show the system 10 as mcludmg a hollow tube 12 havmg a tubular or cyhndπcal design which can be made of anv water impervious deformable mateπal For example, m one embodiment, the hollow tube 12 is a plastic straw havmg accordion-like pleats 14 in the middle region of the hollow tube that permits bending or flexing of the hollow tube Suitable straws mclude NOISY BENDER brand straws and GLAD brand flexible straws which are commercially available Alternatively, other deformable hollow tube matenals which will find use the present system mclude hollow cylinders made of polyethylene, polypropylene, polyesters, polytetrafluoroethylene, nylon, and the like Preferably, the hollow tube has a flexible jomt or is bendable without forming a kink m said hollow tube, or is bent wherem the bend causes a deviation from the longitudinal axis extendmg through the center of said hollow tube is preferably about 90°, but can range from 0° to 180° As further shown Figures 1A-1D, the hollow tube 12 is configured to receive a tissue sample 15 compπsmg filter paper 16 as a receptacle for the tissue sample 17 e g , leaf tissue The tissue sample 17 (a plant leaf) can be placed directly on the filter paper 16 and the paper folded to encompass the sample In a preferred embodiment, the filter paper can mclude a dotted lme 19 to direct a user on placement ofthe fold. The amount of sample 17 placed m the filter paper receptacle will normally be dependent upon a number of factors well recognized by those of ordinary skill in the art, mcludmg the amount of the substance of mterest present in, or the expected extraction efficiency from, the sample Where it is desired to extract nucleic acids from plant leaf samples, about 50-200mg of sample is usually adequate, although the hollow tube 12 can be constructed to accommodate much larger sample sizes Once the sample 14 is prepared and placed mto the filter paper, it can be inserted mto the hollow tube 12, preferably with no tissue sample protrudmg out of the proximal end 13 of the hollow tube 12 Optionally, an eluant buffer can be introduced mto the hollow tube 12 to facilitate efficient recovery and/or the stability of the substance of mterest For example, the filter paper can be soaked with an eluant buffer pnor to insertion mto the hollow tube assembly The filter paper then can serve to provide a reservoir of eluant m immediate contact with the sample The hollow tube 12 is preferably constructed of a mateπal which is impermeable to the substance of mterest This serves to protect the remainder of the system from bemg contaminated with the extracted substance, and to prevent the substance from bemg lost pnor to completing the compression phase ofthe method It follows that if an eluant buffer is used, that the hollow tube 12 should preferably be impermeable to the buffer as well as the substance of mterest In a preferred embodiment, the tissue sample is placed on a filter paper 16 pπor to insertion ofthe sample mto the hollow tube 12 The filter paper 16 can facilitate loading ofthe sample mto the hollow tube, or can prevent the tissue sample from bemg squeezed out of the hollow tube 12 duπng compression of the tube and sample usmg the compression means As descnbed herem, the compression of the hollow tube and sample is referred to as the "extraction step" The present hollow tube must have at least one deformable region wherem the sample can be disposed. The rest ofthe hollow tube may be flexible or rigid, provided that it does not interfere with the ability ofthe compressmg means to compress the deformable region of the hollow tube and express the substance of mterest from a sample disposed withm the hollow tube Another embodiment ofthe subject mvention concerns a hollow tube assembly havmg a liner disposed therem or integral therewith for retaining debns from a sample, while permitting the liquid and substance of mterest extracted from the sample to pass therethrough The lmer, which can be filter mateπal, mcludmg 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 descnbed heremabove An advantage of the hollow tube assembly havmg a liner disposed therem is that the sample can be simply placed in the hollow tube without pπor preparation of a filter paper The hollow tube assembly havmg a lmer disposed therem is preferably a deformable matenal as descnbed herem for other embodiments of the hollow tube In a preferred embodiment, the hollow tube assembly can be substantially a flattened conical shape, forming a funnel-shaped receptacle for receivmg the biological sample, and having a liner which substantially covers the inner wall of the tube assembly and is closed at its vertex to prevent retained sample debns from mixing with the liquid or substance of mterest extracted from the sample The corneal hollow tube assembly can have open or closed ends, but preferably has at least one open end, and more preferably, has a top end (the wider end of he cone) that is open for receiving the sample In a most preferred embodiment, the hollow tube assembly has a top end and a bottom end, both of which are open In one embodiment, the conical hollow tube assembly can be formed by sealmg together two or more substantially tnangular pieces of a liquid-impermeable matenal to form a comcal hollow tube assembly The pieces ofthe comcal hollow tube assembly can be adhered by any known hquid- lmpermeable adhesive or can be of a mateπal that adheres to itself when heated, such as melting together at the pomts where heat is applied, thereby forming a heat-seal In a further embodiment, where the hollow tube is formed by adheπng together more than one piece of matenal, at least one of said pieces of matenal 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 identifier placed or stamped thereon to serve as a marker or identifier ofthe sample source Preferably, the tag is detachable from the hollow tube assembly, e g , by perforations allowing the tag to be easily detached along the perforation lme The label or identifier affixed thereto can be matched to a second, correspondmg label affixed to the body of the comcal hollow tube assembly In a most preferred embodiment, the conical hollow tube assembly and the detachable tag have matching bar¬ code labels for identifying the sample source and sample For example, m practicing the mvention using this embodiment ofthe subject apparatus, a sample collector obtainmg a substance of mterest from a plant can identify an appropπate plant from which the sample is to be taken, remove a leaf sample from the source plant, and place it in the comcal hollow tube assembly, detach the perforated tag and attach it to the source plant to mark or label that plant consistent with the sample taken therefrom The comcal hollow tube assembly embodiment is illustrated in Figure 8 Advantageously, by usmg the embodiment of the comcal hollow tube assembly havmg a lmer disposed therem, the assembly can be stored and shipped substantially flat, can be easily transported in the field, can facilitate collection of samples by allowing a collector to open the flat assembly and place a sample directly inside the hollow tube assembly, can facilitate labehng of sample source and sample, and can eliminate the need for separate filter paper which samples are wrapped Further, whereby this comcal hollow tube embodiment can facilitate use m the field, a buffer packet can also be mcluded Preferably, the desired embodiment can mclude a powdered buffer component that can be reconstituted with water, or can be appropπately mixed with the fluid from the sample such that added water for reconstituting the buffer is not needed The present system further compnses a compressmg means which operates m combmation with the present hollow tube to deform the region contaimng the sample, thereby compressmg the sample and expellmg therefrom at least a portion ofthe substance of mterest One embodiment of a compressmg means, e g., a pair of rollers which can engage the hollow tube m a manner so as to compress the hollow tube and a sample disposed therem, is shown in Figure 2 More specifically, the present device 20 shown in Figure 2 can mclude a pair of rotatable rollers 21 and 22 juxtaposed 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 facilitating the rollers' counter-rotation At least one ofthe gears 24 can be connected directly or indirectly to a dnving means (not shown) which dnves the rollers 21 and 22 The dπvmg means, which can be mcluded as part of the subject mvention, can be adapted to be motoπzed and, for example, to run at vaπous speeds and m different directions, as the hollow tube 12 translates between rollers 21 and 22 Alternatively , the dnving means can be a hand-crank or battery-dπven mechanism useful in field applications where electricity may not be available In addition, it is considered routme to provide a feature for adjusting the pressure exerted between the rollers, and to control such pressure automatically to, for example, maintain constant pressure as the portion ofthe hollow tube havmg sample disposed therem passes between the rollers 21 and 22 Rollers 21 and 22 are preferably made of a πgid matenal such as hard plastic or metal However, m certam applications, rollers of a firm, slightly deformable matenal such as hard rubber may be more effective as would be apparent to one skilled m the art m view of the teachings herem In the normal operation of device 20, rollers 21 and 22 initially contact each other. As the hollow tube 12 translates between the rollers, they remain m substantial contact, except m the region where their contacting relationship is interrupted by the translating hollow tube 12 passing therebetween In an altemate embodiment, rollers 21 and 22 can be positioned to have a gap between them, and die system will preferably be adapted to allow the operator to adjust the gap size An appropπate gap dimension is selected depending on the particular sample and hollow tube matenal As the sample-containing area 26 ofthe hollow tube 12 passes between the rollers 21 and 22, die tissue sample is compressed and fluid containing the substance of mterest flows backwards toward the distal end 25 ofthe hollow tube 12 The fluid can be collected m a collection means 28 that can optionally contain a glass or paper filter element 29 Vaπous other column packing mateπals can be used as well depending on the substance of mterest The bottom end 27 of the collection means 28 can be adapted to eliminate the collected fluid For example, the bottom end can be open or can be stopped or fitted with a valve m order to allow an operator to discard the fluid as desired If the substance of mterest remains on the filter element 29 then the fluid is discarded If the substance of mterest remains solubilized m the fluid, then the fluid can be collected or retained for further punfication or analysis Figure 3 A shows a collection means or collection tube 30 that has a top end 31 , that is open to receive the fluid from the hollow tube after the sample is compressed, and a tapered, bottom end 32 that can be closed In a preferred embodiment, the closed bottom end can be scored to facilitate breaking off the closed end at a desired time to form an open bottom end of the collection tube Optionally, a filter element 33 can be positioned toward the tapered end The filter element 33 can be filter paper or glass fibers or the like compacted mto the tapered end 32 or preferably held mto place by an O-nng 34 Altematively, as illustrated m FIG 3B the filter element 33 can be held mto place by porous polyethylene discs 34 and 36 The plant source of an individual sample, the hollow tube (straw) and the collection tube can be labelled, e g , bar-coded, for audit and research purposes Such labelling can make it easy to identify plants or can facilitate matching the sample or source plant with the analytical results Figure 4, a photograph, shows a preferred embodiment of the present invention in a procedure to isolate plant nucleic acids from leaf tissue One end 40 of a flexible straw is loaded with a leaf sample contamed in a folded filter paper that was wetted with a nucleic acid extraction solution The loaded end of the straw 40 is run through the compressmg means, namely, the rollers 42 and 43, which are dπven by an electric motor (not shown) Intracellular plant fluid is expressed or extracted from the leaf sample and exit the hollow tube at the distal end 44 mto a collection cube tube 45 that contams a Whatman glass fiber filter 46 held mto place by porous polyethylene discs above and below it The collected fluid can optionally be mcubated at a temperature elevated above or lowered below room temperature, which is typically accepted by the ordmanly skilled artisan to be about 25 °C The nucleic acids m the expressed fluid are then precipitated by addmg an alcohol, e g , ethanol, or an adjacent homologue or positional isomer thereof The fluid is then mixed and allowed to stand for about 10 minutes The fluid is discarded by breaking off the scored tip ofthe tapered end ofthe collection cube tube or poking a hole in the tapered end Fluid is forced through the filter element by centrifugal force, the application of pressure to the open end, or the application of vacuum to the punctured or otherwise opened end The filter element is then washed with an alcohol wash solution The precipitated nucleic acids are then redissolved with a warm aqueous buffer and eluted from the filter The substantially pure nucleic acids are then analyzed employmg standard techniques FIG 5, a photograph, shows a 96 well contamer or mega titre tray 51 that contams two collection cube tubes 52 and 53 The cube tubes 52 and 53 fit snugly mto the tray 50 and the whole umt can be spun m a centπfuge to spm off liquid from the bottoms ofthe cube tubes 52 and 53 FIG. 6, a photograph, shows an empty collection cube tube 61 with an open top end 62 and a closed bottom end 63 A filter paper 64 havmg a diameter of approximately the diameter ofthe inner wall of the cube tube towards its closed end 63 is sandwiched between two discs of porous polyethylene 65 and 66. The sandwiched 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 conducting the present extraction process, the liquid remains above the sandwiched filter element. The liquid will pass through the filter paper when the cube tube is centrifuged or when pressure or vacuum is applied. The bottom end ofthe cube tube can be opened, as described, to allow the liquid to be collected, if desired, or discarded if not needed. FIG. 7, a photograph, depicts an extraction kit (minus buffers) comprising a hollow tube, e.g., flexible plastic straw 71, a filter paper 72 and a loaded collection cube tube 67. A ruler is shown on the left side ofthe photo to give an idea of the size of typical kit components. Buffers are added and will depend on the sample and the substance of interest desired to be extracted. FIG. 8 is a photograph ofthe conical hollow tube embodiment showing the detachable label and corresponding label on the body ofthe hollow tube. The embodiment shown is open at both top and bottom ends. The following examples serve to illustrate certain preferred embodiments and aspects ofthe present invention and are not to be construed as limiting the scope thereof. Experimental In the experimental disclosure which follows, all weights are given in grams (g), milligrams (mg), m-crograms (μg) or moles (mol). All concentrations are given as percent by volume (%), molar (M), millimolar (mM) or micromolar (μM). All volumes are given in liters (L), milliliters (mL) or microliters (μL), unless otherwise indicated. The following examples demonstrate the operation ofthe system ofthe present invention to extract DNA and RNA from a plant leaf sample and should not be construed as limiting its scope. Example 1 : Solutions The following buffer solutions were employed: Nucleic Acid Extraction Buffer: 100 mM Tris-HCl(pH 8.0), 10 mM Ethylenediaminetetraacetic acid (EDTA), 10 mM ethylene glycol-bis(BiJ-an-inoethyl)N,N,N-tetracetic acid, 10 mM 1,10-ortho- phenanthroline, 1.5% polyvinylpyrolidone 40, 1% sodium dodecyl sulfate and 10 ug/ml proteinase K. 10 M ammonium acetate 100% Isopropanol Ethanol wash 70% ethanol, 1 M ammomum acetate RNAse Ethanol wash 70% ethanol, 1 mM ammomum acetate, 0 2 ug ml DNAse-free RNAse A TE Storage Buffer lOmM Tπs-HCI (pH 8 0), 0 1 mM EDTA Example 2 Plant DNA Extraction A hollow tube was prepared by cuttmg the long end of a flexible plastic straw (GLAD brand) about 2 5 inches from the accordion-like pleats A maize leaf sample of about 1-1/2 cm x 10cm was folded and placed on a 3mm Whatman filter paper The filter paper was folded lengthwise with the leaf tissue on the inside The folded paper was then inserted mto the end ofthe hollow tube bemg careful that no leaf tissue was protrudmg out of the end of the hollow tube DNA extraction buffer (500 μL) 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 honzontal when addmg the DNA extraction buffer to keep the buffer m close proximity to the plant tissue This end was then compressed by passmg it through a roller assembly as shown in Figures 2 and 4 The expressed sample was collected in the specially adapted tube and incubated for 30 mmutes to allow complete protem digestion and nucleic acid solubilization The sample can be stored indefinitely in this form if kept in the dark The sample was adjusted to 2 5 mM ammomum acetate by the addition of one third volume of 10 mM ammomum acetate and then precipitated with the addition of an equal volume of isopropanol The tube was punctured and the precipitated nucleic acids were captured on the Whatman GFB filter paper as the solution was centnfuged through the filter The filter was washed 3 times with an ethanol wash (descnbed m Example 1 ) Total nucleic acids (l e , DNA and RNA) were eluted with the filter with hot (65 °C) TE storage buffer RNA free of DNA or, conversely DNA free of RNA, can be prepared from the total nucleic acid sample by means well known to one of ordinary skill m the art One means to make RNA-free DNAse is to wash the filter with an RNAse/Ethanol wash pπor to elution of the DNA In order to verify that the extraction method was successful, the DNA precipitates are dissolved m a DNA storage buffer (lOmM Tns-HCl, pH 8 0, lOmM NaCl, ImM EDTA) The quantities of DNA or RNA extracted may be determined by methods well known m the art The quality of the extracted DNA can be determined by size (e g , >50,000bp) and by its sensitivity to vaπous restnction endonucleases under standard conditions The quality of RNA may be determined by absence of degradation and other means well known m the art The description of the preferred embodiments is not mtended to limit the scope of the claimed mvention It will be evident from the followmg claims that the claimed mvention mcludes modifications which would be obvious to a person of ordmary skill m the art One can readily see that the present mvention avoids a lot of transfer steps and contamination of hardware The present mvention is basically a one tube process that serves as a field collection tube and punfication system that decreases contammation and reduces the chance for mislabellmg errors

Claims: Claims 1 A system for extracting a substance from a biological sample, said system compπsmg (a) a hollow tube assembly havmg a deformable region, and (b) means for collection of the extracted substance 2 The system, accordmg to claim 1, wherem said hollow tube assembly compnses a hquid- impermeable matenal 3 The system, according to claim 2, wherem said liquid-impermeable matenal is a polymer 4 The system, according to claim 1 , wherem said hollow tube assembly has a bendable jomt capable of forming, or is bent to form, an angled hollow tube 5 The system, accordmg to claim 1 , wherem said hollow tube is comcal 6 The system, accordmg to claim 5, wherein said comcal hollow tube has disposed therem a filter as a lmer of an inner wall of said hollow tube 7 The system- accordmg to claim 6, wherem said filter lmer is mtegral with the inner wall of said hollow tube 8 The system, accordmg to claim 1, wherem said collection means is a collection tube 9 The system, accordmg to claim 1 , wherem said collection means further compnses a filter element disposed therem 10 The system, accordmg to claim 9, wherein said filter element is a filter disc 1 1 The system, accordmg to claim 9, wherem said filter element is held withm said collection tube by an O-nng 12 The system, accordmg to claim 9, wherem said filter element is held withm said collection tube by bemg sandwiched between two porous polyethylene discs 13. The system, according to claim 1, wherem said system further comprises a compressmg means 14 The system, accordmg to claim 13, wherem said compressmg means compnses at least a pair of rollers which are engaged at sufficient pressure to compress the biological sample to express liquid therefrom, while allowmg a hollow tube assembly to deformably pass therebetween 15 The system, accordmg to claim 13, wherem said compressmg means is motor-dπven 16 A method of extracting a substance of mterest from a biological sample, said method compnsmg the steps of a placing a sample compnsmg the substance of mterest m a deformable region of a hollow tube assembly, b deforming the hollow tube assembly m the deformable region that contains the sample whereby a liquid compπsmg the substance of mterest is expressed from the sample, and c collecting the expressed qmd 17 The method, accordmg to claim 16, wherem said substance of mterest is selected from the group consistmg of a nucleic acid or an ammo acid 18 The method, accordmg to claim 17, wherem said nucleic acid is DNA or RNA 19 The method, accordmg to claim 1, wherem said biological sample is a plant tissue 20 The method, accordmg to claim 16, wherem said defoiming step (b) employs a pair of engaging rollers 21 The method, accordmg to claim 16, wherem said method further compnses wτappιng said biological sample filter paper pπor to placing the sample m said hollow tube assembly. 22 The method, accordmg to claim 21 , wherem said filter paper is soaked with an elution buffer 23 An extraction kit which compnses a a hollow tube assembly that is deformable at least at one end and which is adapted to receive a sample contaimng a substance of mterest, b a means for collectmg the substance of mterest from said sample, and c a buffer composition capable of stabdizmg, extracting or punfymg the substance of mterest 24 The extraction kit, accordmg to claim 23, further compπsmg a filter paper to hold the sample and wherem the collectmg means (b) is a collection tube compπsmg an open end, a closed end, and a filter element or column packing

Inventors: MURRAY MICHAEL, MURRAY, MICHAEL
Applicants: MYCOGEN PLANT SCIENCES
Application year: 1996
Main classification: C12M1
IPC codes: C12M1, C12M3, C12N15, C12Q1, G01N1