WO1999042620A2 - Articles of manufacture and methods for staining biomolecules - Google Patents
Articles of manufacture and methods for staining biomolecules Download PDFInfo
- Publication number
- WO1999042620A2 WO1999042620A2 PCT/US1999/003704 US9903704W WO9942620A2 WO 1999042620 A2 WO1999042620 A2 WO 1999042620A2 US 9903704 W US9903704 W US 9903704W WO 9942620 A2 WO9942620 A2 WO 9942620A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- article
- backing
- biomolecules
- indicator
- manufacture according
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 31
- 238000010186 staining Methods 0.000 title claims description 29
- 239000011248 coating agent Substances 0.000 claims abstract description 46
- 238000000576 coating method Methods 0.000 claims abstract description 46
- 238000002372 labelling Methods 0.000 claims description 14
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical group [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 12
- 229960005542 ethidium bromide Drugs 0.000 claims description 12
- 230000002285 radioactive effect Effects 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 108091034117 Oligonucleotide Proteins 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 239000003068 molecular probe Substances 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 8
- 239000003292 glue Substances 0.000 claims description 8
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 7
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 7
- 229920000936 Agarose Polymers 0.000 claims description 6
- -1 basic red 49 Chemical compound 0.000 claims description 6
- 229920002472 Starch Polymers 0.000 claims description 5
- 229920002401 polyacrylamide Polymers 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 4
- 239000001913 cellulose Substances 0.000 claims description 4
- 238000009792 diffusion process Methods 0.000 claims description 4
- 235000013312 flour Nutrition 0.000 claims description 4
- SCMDRBZEIUMBBQ-UHFFFAOYSA-N (1e)-1-[(8-amino-3,7-dimethyl-10-phenylphenazin-10-ium-2-yl)hydrazinylidene]naphthalen-2-one;chloride Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N\N=C\3C4=CC=CC=C4C=CC/3=O)C=C2[N+]=1C1=CC=CC=C1 SCMDRBZEIUMBBQ-UHFFFAOYSA-N 0.000 claims description 3
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 claims description 3
- QAMCXJOYXRSXDU-UHFFFAOYSA-N 2,4-dimethoxy-n-[2-(1,3,3-trimethylindol-1-ium-2-yl)ethenyl]aniline;chloride Chemical compound [Cl-].COC1=CC(OC)=CC=C1NC=CC1=[N+](C)C2=CC=CC=C2C1(C)C QAMCXJOYXRSXDU-UHFFFAOYSA-N 0.000 claims description 3
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 3
- POELEEGOWIJNBI-UHFFFAOYSA-N 3-[2-[[4-(diethylamino)phenyl]diazenyl]-6-ethoxy-1,3-benzothiazol-3-ium-3-yl]propanamide;chloride Chemical compound [Cl-].S1C2=CC(OCC)=CC=C2[N+](CCC(N)=O)=C1N=NC1=CC=C(N(CC)CC)C=C1 POELEEGOWIJNBI-UHFFFAOYSA-N 0.000 claims description 3
- XXACTDWGHQXLGW-UHFFFAOYSA-M Janus Green B chloride Chemical compound [Cl-].C12=CC(N(CC)CC)=CC=C2N=C2C=CC(\N=N\C=3C=CC(=CC=3)N(C)C)=CC2=[N+]1C1=CC=CC=C1 XXACTDWGHQXLGW-UHFFFAOYSA-M 0.000 claims description 3
- 108010076830 Thionins Proteins 0.000 claims description 3
- MPBRYMWMMKKRGC-UHFFFAOYSA-M carbocyanin DBTC Chemical compound [Br-].C1=CC=CC2=C([N+](=C(C=C(C)C=C3N(C4=C5C=CC=CC5=CC=C4S3)CC)S3)CC)C3=CC=C21 MPBRYMWMMKKRGC-UHFFFAOYSA-M 0.000 claims description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 3
- 239000013078 crystal Substances 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- DWCZIOOZPIDHAB-UHFFFAOYSA-L methyl green Chemical compound [Cl-].[Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)[N+](C)(C)C)=C1C=CC(=[N+](C)C)C=C1 DWCZIOOZPIDHAB-UHFFFAOYSA-L 0.000 claims description 3
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 3
- XJCPMUIIBDVFDM-UHFFFAOYSA-M nile blue A Chemical compound [Cl-].C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4[O+]=C3C=C(N)C2=C1 XJCPMUIIBDVFDM-UHFFFAOYSA-M 0.000 claims description 3
- INAAIJLSXJJHOZ-UHFFFAOYSA-N pibenzimol Chemical compound C1CN(C)CCN1C1=CC=C(N=C(N2)C=3C=C4NC(=NC4=CC=3)C=3C=CC(O)=CC=3)C2=C1 INAAIJLSXJJHOZ-UHFFFAOYSA-N 0.000 claims description 3
- QWYZFXLSWMXLDM-UHFFFAOYSA-M pinacyanol iodide Chemical compound [I-].C1=CC2=CC=CC=C2N(CC)C1=CC=CC1=CC=C(C=CC=C2)C2=[N+]1CC QWYZFXLSWMXLDM-UHFFFAOYSA-M 0.000 claims description 3
- INCIMLINXXICKS-UHFFFAOYSA-M pyronin Y Chemical compound [Cl-].C1=CC(=[N+](C)C)C=C2OC3=CC(N(C)C)=CC=C3C=C21 INCIMLINXXICKS-UHFFFAOYSA-M 0.000 claims description 3
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 claims description 3
- 229920001059 synthetic polymer Polymers 0.000 claims description 3
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 3
- 239000000499 gel Substances 0.000 abstract description 29
- 239000012192 staining solution Substances 0.000 abstract description 7
- 239000012528 membrane Substances 0.000 abstract description 6
- 231100000331 toxic Toxicity 0.000 abstract description 5
- 230000002588 toxic effect Effects 0.000 abstract description 5
- 239000000383 hazardous chemical Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 41
- 239000000523 sample Substances 0.000 description 15
- 239000000872 buffer Substances 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229920000881 Modified starch Polymers 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 239000004744 fabric Substances 0.000 description 3
- 235000019426 modified starch Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000004368 Modified starch Substances 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000002894 chemical waste Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000007873 sieving Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- 238000012800 visualization Methods 0.000 description 2
- AGNTUZCMJBTHOG-UHFFFAOYSA-N 3-[3-(2,3-dihydroxypropoxy)-2-hydroxypropoxy]propane-1,2-diol Chemical compound OCC(O)COCC(O)COCC(O)CO AGNTUZCMJBTHOG-UHFFFAOYSA-N 0.000 description 1
- 235000007173 Abies balsamea Nutrition 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 239000004857 Balsam Substances 0.000 description 1
- 229920000298 Cellophane Polymers 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 244000018716 Impatiens biflora Species 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920000297 Rayon Polymers 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002964 rayon Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Definitions
- Electrophoresis techniques have become principal tools for characterizing biomolecules.
- the method is based on the fact that macromolecules such as DNA, RNA and proteins possess a charge and can therefore move in an electric field through sieving materials such as agarose or poly aery la ide.
- the application of electrophoretic techniques has required the development of chemical indicators for use in visualizing separated macromolecules.
- One such indicator used to visualize DNA is ethidium bromide (EtBr).
- EtBr ethidium bromide
- the disadvantage of this widely used stain is that it is a potent mutagen.
- the potential personal hazard of directly contacting such a solution and the environmental hazard of pouring it or other hazardous chemicals down the drain has resulted in a need for a better and safer method of preparing, using and disposing of such toxic solutions.
- an object of the present invention to provide a readily manufacturable apparatus capable of safely staining or labeling biomolecules.
- an article of manufacture comprising a flexible backing and a dry coating adhered to a surface of the backing, wherein the coating comprises an indicator that stains or labels a biomolecule.
- the backing promotes good contact with a gel containing a biomolecule, is impermeable, allows the coating solution to be evenly spread and dried and is easily dispensable.
- the coating solution is selected from the group consisting of starch, glue, gum, flour, agarose or polyacrylamide, or mixtures of two or more of these.
- the indicator is selected from the group consisting of stains, dyes or labeled probes.
- a method for staining or labeling biomolecules comprising applying an article of manufacture comprising a flexible backing and a dry coating adhered to a surface of the backing, wherein the coating comprises an indicator that stains or labels a biomolecule, to a gel or support containing the biomolecules for a sufficient period of time to allow diffusion to the biomolecules.
- kits suitable for use in a method for staining or labeling biomolecules comprising (A) an article of manufacture comprising a flexible backing and a dry coating adhered to a surface of the backing, wherein the coating comprises an indicator that stains or labels a biomolecule; (B) reagents to effect staining or labeling of the biomolecules; and (C) instruments to effect staining or labeling of the biomolecules.
- Figure 1 Demonstration of EtBr staining of DNA using two types coated backings.
- Figure 1(a) shows an agarose gel stained with a backing comprising White Uncoated Paper #100.
- Figure 1(b) shows an agarose gel stained with a backing comprising Prevail Paper #125. The gels were stained for 2-3 minutes and then placed on a Short Wave UV Transilluminator for visualization of the DNA bands and for obtaining photographs.
- the present invention provides an article of manufacture and methods for safely staining or labeling biomolecules.
- the stable, resilient and pliable article can be used repeatedly in a method for staining biomolecules in gels and membranes without directly contacting toxic staining solutions and without handling delicate gels which contain such stains.
- the process eliminates the environmental hazard resulting from the disposal of toxic stains since the backing with the stain can be disposed of in a solid chemical waste container.
- the process reduces the amount of stain to be used and eliminates the personal hazards associated with staining biomolecules since the operator will no longer have direct contact with the stain itself.
- the article is easily manufactured which reduces the cost of the article and ensures its accessibility in the marketplace.
- an article of manufacture which comprises a flexible backing and a dry coating adhered to a surface of the backing, wherein the coating comprises an indicator that stains or labels a biomolecule.
- the backing is flexible, promotes good contact with a support or gel containing a biomolecule, is impermeable and allows the coating solution to be evenly spread and dried.
- the backing can be composed of cellulose-based compounds, such as various papers, for example wall paper, cloth or vinyl, 3M WhatmanTM paper, vinyl paper, cloth paper, prepasted paper, to name a few.
- the backing can be any wood, preferably light and strong as Balsam of about 1/16-1/8 of an inch in thickness, any glass such as borosilicate of about 50 mm, any cloth, cotton, rayon, tafetta, mesh, wool, synthetic polymers such as nylon, acetate, mixtures of cotton and acetate, polypropylene or polyethylene, polycarbonate, acrylic, cellophane acetate, Gelbond (FMC Bioproducts, Maine) to name a few, and any sponge.
- the backing is comprised of two layers, consisting of a permeable layer and an impermeable layer.
- the coating solution comprises an adhering solution and an indicator capable of staining or labeling a biomolecule.
- Any solution which adheres to the backing and is preferably amenable to drying and rewetting, and does not degrade the biomolecule to be stained or the gel or support which retains the biomolecule to be stained can be utilized.
- adhering solutions comprised of modified starches and their derivatives. Starch solutions can be modified in a variety of ways, including digestion with amylase.
- Other examples of adhering solutions include, but are not limited to, water-based glues, flour, agarose, polyacrylamide and its derivatives, and any mixture there of.
- glues include, but are not limited to, wall-paper glue, paper glue and multiple function glue.
- Gums such as locust bean gum, also can be used. Adhering solutions containing silica or diatomaceous earth also are advantageous.
- one or more indicators are entrapped in the adhering solution.
- the desired indicator is mixed with the coating solution at varying amounts depending on the sensitivity of the stain.
- the indicator can be in the form of a solution.
- the indicator can be dissolved in water or a buffer. Suitable buffers can comprise mixtures of aqueous and organic solutions or comprise only organic solutions, such as isopropanol for ethidium bromide.
- the indicator can be mixed into the coating solution as a powder.
- the stain is spread onto a backing onto which a coating solution has been dried and which is capable of being rewetted again and redried.
- indicators can be utilized individually or in combination in the present invention to stain or label biomolecules.
- Preferred indicators include, but are not limited to, stains, dyes and labeled probes. Examples of acceptable stains and dyes include, but are not limited to, ethidium bromide, YoYo (Molecular Probes, Inc., Boulder, CO), Toto (Molecular Probes, inc.
- the indicator comprises one or more probes complementary to the biomolecule to be detected.
- a complementary probe include, but are not limited to, a labeled synthetic oligonucleotide, an antibody and antibody fragment.
- probes can be added to the coating solution, or applied to the dried backing with the coating solution instead of, or in addition to, a stain.
- the probe can be labeled with a radioactive label or a non-radioactive label such as biotin, alkaline phosphatase, and horseradish peroxidase, or a chemiluminescent reagent, among others.
- the labeled probe can be added at a concentration of about 1 - 1000 pmol/ml, preferably about 100 pmol/ml.
- compositions are added to the coating solution.
- Such compositions for example tris acetate, polyethylene glycol and its derivatives, and triglycerol, can facilitate staining or can stabilize the stain or biomolecules.
- Incorporating a hybridization buffer into the coating solution is especially useful when the indicator comprises a labeled probe.
- the coating solution with an indicator can be spread onto one or both sides of the backing.
- the backing can be spread with the coating solution, dried, and then coated with an indicator solution.
- Coating and indicator solutions can be applied onto the backing by rolling them with a sponge roller or automated roller, or by brashing or spraying them onto the backing.
- the coated backing can be air dried, oven dried, or dried in a microwave oven.
- a method for staining biomolecules comprising applying an article of manufacture comprising a flexible backing and a dry coating adhered to a surface of the backing, wherein the coating comprises an indicator that stains or labels a biomolecule, to a gel or support containing the biomolecules for a sufficient period of time to allow diffusion to the biomolecules.
- the coating comprises an indicator that stains or labels a biomolecule, to a gel or support containing the biomolecules for a sufficient period of time to allow diffusion to the biomolecules.
- Usually, about 0.5 minute or longer of direct contact is required to allow the indicator to diffuse to the biomolecules.
- biomolecules include, but are not limited to, DNA, RNA and proteins.
- the present invention is used to stain or label biomolecules contained in gels.
- a gel containing biomolecules can be made of any sieving material such as agarose, starch, polyacrylamide synthetic matrices, or various blends of matrices.
- the backing is peeled off, and can be disposed of in the solid chemical waste, or reused.
- a fresh application of the desired stain may be necessary upon reuse. This process prevents direct contact with toxic staining solutions, minimizes the volume of the staining solution utilized and saves the environment from any hazards which such a staining solution may produce.
- the present invention is used to label biomolecules immobilized on a support.
- Hybridization assays such as Southern, northern and western assays, utilize labeled probes to detect specific DNA, RNA or protein species immobilized onto membranes, such as nylon or nitrocellulose.
- immobilized biomolecules may be detected using the method described above.
- a probe for example a labeled synthetic oligonucleotide complementary to the biomolecule to be detected, is added to the coating solution, or applied to the dried backing with the coating solution.
- the oligonucleotide can be labeled with a radioactive label or a non-radioactive label such as biotin, alkaline phosphatase, horseradish peroxidase or a chemiluminescent reagent among others.
- a radioactive label or a non-radioactive label such as biotin, alkaline phosphatase, horseradish peroxidase or a chemiluminescent reagent among others.
- the backing containing the labeled probe is applied to the membrane such that the probe is in direct contact with the support containing the biomolecules to be detected and even transfer of the probe from the backing to the biomolecules is effected.
- the backing and the support are left in contact for several minutes up to eighteen hours or overnight. Detection of complementary binding between the labeled probe and the immobilized biomolecule may be carried out by methods known in the art.
- the present invention provides a kit suitable for use in a method for staining or labeling biomolecules, the kit comprising an article of manufacture comprising a flexible backing and a dry coating adhered to a surface of the backing, wherein the coating comprises an indicator that stains or labels a biomolecule, reagents to effect staining or labeling of the biomolecules, and instruments to effect staining or labeling of the biomolecules.
- the kit can be an educational or research kit, whereby other reagents for the preparation of the separation gels are included, as well as other reagents which facilitate detecting stained biomolecules, for example buffers, glycerol solutions or enzymes.
- Instruments which facilitate staining or detecting biomolecules can be included in the kit.
- a roller which can be used to promote effective contact between the backing and the gel or support containing the biomolecules can be included.
- a cassette into which the backing and the gel or support are placed and which promotes effective contact between the two can be included in the kit.
- a spray applicator which can be used to apply reagents effecting staining can be included.
- a kit for staining biomolecules may be packaged in a variety of ways.
- the coated backing When the kit contains a backing with a coating solution comprising ethidium bromide for staining DNA or RNA, the coated backing must be stored such that it is not exposed to light, since ethidium bromide is light sensitive.
- a preferred package for this embodiment is a foil pouch. It is contemplated that a plurality of backing strips can be stored in a roll such that the desired size of backing can be cut away either using scissors or using a sharp serrated metal edge adhered to the box in which the roll is stored. Additionally, the use of perforated paper can simplify the dispensing process. In these forms, it is preferable that the coated backing be covered with a liner which when peeled away exposes the coating just prior to use so that the backings in the roll do not adhere to each other.
- Such a liner can be made from paper or other release liner.
- the backing can be stacked in sheets, folded and stored in a dispenser for easy removal of one sheet at a time.
- the coating solution can be supplied in a separate container for application onto the backing when needed.
- the coating solution can be supplied with or without stain, and the staining solution or powder can be provided in yet another container.
- An adhering solution is prepared by forming a 10% solution of modified starch using warm, distilled water. An appropriate amount of ethidium bromide is then added to the adhering solution, preferably to a final concentration of between 0.05 mg/ml and 0.2 mg/ml. The resulting solution is spread over one side of a cellulose-based paper using a sponge roller and allowed to dry. After the coating solution has dried, the paper may be rolled up and stored until use.
- Ethidium bromide was added to a final concentration of 0.0625 mg/ml.
- the resulting coating solution was spread onto two types of paper backing, White Uncoated Paper #100 and Prevail Paper #125, using a sponge roller and allowed to air dry in the dark. Approximately 0.5 ml of the coating solution was applied to each backing. Following fractionation, the gels were inverted, and the coated backing was placed on the gel such that the ethidium bromide was in contact with the gel containing the DNA. The gels were stained for 2-3 minutes and then placed on a Short Wave Ultraviolet Transilluminator for visualization of the DNA bands.
- the results in Figure 1 show that the present invention provides a safe and effective means of staining DNA molecules contained in gels.
- Figure 1(a) is a photograph of a gel stained with the coated backing comprising White Uncoated Paper #100.
- Figure 1(b) is a photograph of a gel stained with the coated backing comprising Prevail Paper #125.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99908309A EP1057001A2 (en) | 1998-02-19 | 1999-02-19 | Articles of manufacture and methods for staining biomolecules |
CA002321100A CA2321100A1 (en) | 1998-02-19 | 1999-02-19 | Articles of manufacture and methods for staining biomolecules |
AU27771/99A AU2777199A (en) | 1998-02-19 | 1999-02-19 | Articles of manufacture and methods for staining biomolecules |
JP2000532558A JP2002504351A (en) | 1998-02-19 | 1999-02-19 | Biomolecule staining product and method |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US7517398P | 1998-02-19 | 1998-02-19 | |
US60/075,173 | 1998-02-19 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO1999042620A2 true WO1999042620A2 (en) | 1999-08-26 |
WO1999042620A3 WO1999042620A3 (en) | 1999-11-18 |
WO1999042620A9 WO1999042620A9 (en) | 2000-03-02 |
Family
ID=22124035
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/003704 WO1999042620A2 (en) | 1998-02-19 | 1999-02-19 | Articles of manufacture and methods for staining biomolecules |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1057001A2 (en) |
JP (1) | JP2002504351A (en) |
AU (1) | AU2777199A (en) |
CA (1) | CA2321100A1 (en) |
WO (1) | WO1999042620A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005033342A1 (en) | 2003-09-30 | 2005-04-14 | Molecular Probes, Inc. | Detection of immobilized nucleic acid |
WO2007010532A2 (en) * | 2005-07-18 | 2007-01-25 | Shmuel Bukshpan | Compositions for one step colorimetric analysis and methods of using same |
Citations (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3678151A (en) * | 1969-07-25 | 1972-07-18 | Gugol Clini Tex Inc | Biological staining method |
DE2739008A1 (en) * | 1977-08-30 | 1979-03-15 | Behringwerke Ag | Indicator sheet for use with liquids, esp. blood and urine - having coating of hydrophilic swellable polymer particles, preventing indicator running |
US4224277A (en) * | 1975-04-11 | 1980-09-23 | Boehringer Mannheim Gmbh | Coated slides and apparatus for coating same |
EP0119622A2 (en) * | 1983-03-17 | 1984-09-26 | Fuji Photo Film Co., Ltd. | Integral element for biological reaction and process for the preparation thereof |
WO1987003965A1 (en) * | 1985-12-18 | 1987-07-02 | Celldynamics Ag | Multi-indicator test strips for immuno-assays, their production and use |
EP0246505A2 (en) * | 1986-05-16 | 1987-11-25 | Miles Inc. | Process for the production of test strips by casting method |
EP0274911A1 (en) * | 1987-01-12 | 1988-07-20 | Pall Corporation | Diagnostic device, method for making, and method for using |
US4839297A (en) * | 1986-11-12 | 1989-06-13 | Boehringer Mannheim Gmbh | Test carrier and method for the analytical determination of a component of a body fluid |
EP0320842A2 (en) * | 1987-12-18 | 1989-06-21 | W.R. Grace & Co.-Conn. | Method for binding biological reagents to porous supports |
WO1990008840A1 (en) * | 1989-02-03 | 1990-08-09 | Eastman Kodak Company | Nucleic acid test article and its use to detect a predetermined nucleic acid |
EP0387696A2 (en) * | 1989-03-17 | 1990-09-19 | Abbott Laboratories | Method and device for improved reaction kinetics in nucleic acid hybridizations |
WO1992007955A1 (en) * | 1990-10-26 | 1992-05-14 | Orion-Yhtymä Oy | Method for evaluating the adequacy of clinical specimens for hybridization assays and kit for performing the evaluation |
US5132439A (en) * | 1990-11-19 | 1992-07-21 | Promega Corporation | Protein staining compositions and methods |
WO1993013224A1 (en) * | 1991-12-23 | 1993-07-08 | Chiron Corporation | Process for immobilizing nucleic acid probes on polystyrene surfaces |
WO1993013405A1 (en) * | 1991-12-23 | 1993-07-08 | Tropix, Inc. | Improved membrane for chemiluminescent blotting applications |
US5316638A (en) * | 1989-09-27 | 1994-05-31 | Peter Jackson | Treatment of carbohydrates |
EP0677589A2 (en) * | 1994-03-31 | 1995-10-18 | Johnson & Johnson Clinical Diagnostics, Inc. | Method, test element and test kit for semi-quantitative detection of target nucleic acid |
US5514501A (en) * | 1994-06-07 | 1996-05-07 | The United States Of America As Represented By The Secretary Of Commerce | Process for UV-photopatterning of thiolate monolayers self-assembled on gold, silver and other substrates |
EP0745689A2 (en) * | 1990-05-11 | 1996-12-04 | Microprobe Corporation | A dipstick for a nucleic acid hybridization assay |
US5585276A (en) * | 1991-05-10 | 1996-12-17 | Wisconsin Alumni Research Foundation | Medium and method for blotting macromolecules |
US5641635A (en) * | 1993-11-12 | 1997-06-24 | Eastman Kodak Company | Dry elements, test devices, test kits and methods for chemiluminescent detection of analytes using peroxidase-labeled reagents |
US5688642A (en) * | 1994-12-01 | 1997-11-18 | The United States Of America As Represented By The Secretary Of The Navy | Selective attachment of nucleic acid molecules to patterned self-assembled surfaces |
US5776684A (en) * | 1995-12-28 | 1998-07-07 | Edvotek | Method for staining biomolecules using a gelled matrix |
-
1999
- 1999-02-19 JP JP2000532558A patent/JP2002504351A/en active Pending
- 1999-02-19 WO PCT/US1999/003704 patent/WO1999042620A2/en active Application Filing
- 1999-02-19 CA CA002321100A patent/CA2321100A1/en not_active Abandoned
- 1999-02-19 EP EP99908309A patent/EP1057001A2/en not_active Withdrawn
- 1999-02-19 AU AU27771/99A patent/AU2777199A/en not_active Abandoned
Patent Citations (23)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3678151A (en) * | 1969-07-25 | 1972-07-18 | Gugol Clini Tex Inc | Biological staining method |
US4224277A (en) * | 1975-04-11 | 1980-09-23 | Boehringer Mannheim Gmbh | Coated slides and apparatus for coating same |
DE2739008A1 (en) * | 1977-08-30 | 1979-03-15 | Behringwerke Ag | Indicator sheet for use with liquids, esp. blood and urine - having coating of hydrophilic swellable polymer particles, preventing indicator running |
EP0119622A2 (en) * | 1983-03-17 | 1984-09-26 | Fuji Photo Film Co., Ltd. | Integral element for biological reaction and process for the preparation thereof |
WO1987003965A1 (en) * | 1985-12-18 | 1987-07-02 | Celldynamics Ag | Multi-indicator test strips for immuno-assays, their production and use |
EP0246505A2 (en) * | 1986-05-16 | 1987-11-25 | Miles Inc. | Process for the production of test strips by casting method |
US4839297A (en) * | 1986-11-12 | 1989-06-13 | Boehringer Mannheim Gmbh | Test carrier and method for the analytical determination of a component of a body fluid |
EP0274911A1 (en) * | 1987-01-12 | 1988-07-20 | Pall Corporation | Diagnostic device, method for making, and method for using |
EP0320842A2 (en) * | 1987-12-18 | 1989-06-21 | W.R. Grace & Co.-Conn. | Method for binding biological reagents to porous supports |
WO1990008840A1 (en) * | 1989-02-03 | 1990-08-09 | Eastman Kodak Company | Nucleic acid test article and its use to detect a predetermined nucleic acid |
EP0387696A2 (en) * | 1989-03-17 | 1990-09-19 | Abbott Laboratories | Method and device for improved reaction kinetics in nucleic acid hybridizations |
US5316638A (en) * | 1989-09-27 | 1994-05-31 | Peter Jackson | Treatment of carbohydrates |
EP0745689A2 (en) * | 1990-05-11 | 1996-12-04 | Microprobe Corporation | A dipstick for a nucleic acid hybridization assay |
WO1992007955A1 (en) * | 1990-10-26 | 1992-05-14 | Orion-Yhtymä Oy | Method for evaluating the adequacy of clinical specimens for hybridization assays and kit for performing the evaluation |
US5132439A (en) * | 1990-11-19 | 1992-07-21 | Promega Corporation | Protein staining compositions and methods |
US5585276A (en) * | 1991-05-10 | 1996-12-17 | Wisconsin Alumni Research Foundation | Medium and method for blotting macromolecules |
WO1993013224A1 (en) * | 1991-12-23 | 1993-07-08 | Chiron Corporation | Process for immobilizing nucleic acid probes on polystyrene surfaces |
WO1993013405A1 (en) * | 1991-12-23 | 1993-07-08 | Tropix, Inc. | Improved membrane for chemiluminescent blotting applications |
US5641635A (en) * | 1993-11-12 | 1997-06-24 | Eastman Kodak Company | Dry elements, test devices, test kits and methods for chemiluminescent detection of analytes using peroxidase-labeled reagents |
EP0677589A2 (en) * | 1994-03-31 | 1995-10-18 | Johnson & Johnson Clinical Diagnostics, Inc. | Method, test element and test kit for semi-quantitative detection of target nucleic acid |
US5514501A (en) * | 1994-06-07 | 1996-05-07 | The United States Of America As Represented By The Secretary Of Commerce | Process for UV-photopatterning of thiolate monolayers self-assembled on gold, silver and other substrates |
US5688642A (en) * | 1994-12-01 | 1997-11-18 | The United States Of America As Represented By The Secretary Of The Navy | Selective attachment of nucleic acid molecules to patterned self-assembled surfaces |
US5776684A (en) * | 1995-12-28 | 1998-07-07 | Edvotek | Method for staining biomolecules using a gelled matrix |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005033342A1 (en) | 2003-09-30 | 2005-04-14 | Molecular Probes, Inc. | Detection of immobilized nucleic acid |
EP1988177A1 (en) | 2003-09-30 | 2008-11-05 | Molecular Probes Inc. | Detection of immobilised nucleic acid |
US7727716B2 (en) | 2003-09-30 | 2010-06-01 | Life Technologies Corporation | Detection of immobilized nucleic acid |
US7977057B2 (en) | 2003-09-30 | 2011-07-12 | Life Technologies Corporation | Detection of immobilized nucleic acid |
US8969004B2 (en) | 2003-09-30 | 2015-03-03 | Life Technologies Corporation | Detection of immobilized nucleic acid |
WO2007010532A2 (en) * | 2005-07-18 | 2007-01-25 | Shmuel Bukshpan | Compositions for one step colorimetric analysis and methods of using same |
WO2007010532A3 (en) * | 2005-07-18 | 2008-01-10 | Shmuel Bukshpan | Compositions for one step colorimetric analysis and methods of using same |
Also Published As
Publication number | Publication date |
---|---|
WO1999042620A9 (en) | 2000-03-02 |
AU2777199A (en) | 1999-09-06 |
JP2002504351A (en) | 2002-02-12 |
EP1057001A2 (en) | 2000-12-06 |
CA2321100A1 (en) | 1999-08-26 |
WO1999042620A3 (en) | 1999-11-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US3975162A (en) | Applying reagent to medium and device therefor | |
US3990850A (en) | Diagnostic test card | |
FI80343B (en) | TESTANORDNING. | |
US20060014298A1 (en) | Method and kitset for sampling and storage of biological samplies | |
JPH024859B2 (en) | ||
JP3078866B2 (en) | Diagnostic test slides and their manufacturing method | |
JP2003530570A (en) | Methods, articles and kits for labeling polymer gels | |
US20010039010A1 (en) | Sample collection medium incorporating material for sample visualization | |
FI78360B (en) | FOERFARANDE FOER FRAMSTAELLNING AV TESTREMSA. | |
JPH06167496A (en) | Dry immunoassay element provided with separative absorption layer | |
CA2480646A1 (en) | Detection of dna-binding proteins | |
US20050230256A1 (en) | Electrophoresis gels with incorporated indicia | |
EP0851228A4 (en) | Highly sensitive fluorescent immunoassay | |
US4883597A (en) | Hydrophobic membrane for drying gel matrices | |
EP1057001A2 (en) | Articles of manufacture and methods for staining biomolecules | |
RU2001109257A (en) | METHOD FOR QUICK SCREENING OF ANALYZED SAMPLES | |
US5776684A (en) | Method for staining biomolecules using a gelled matrix | |
JPH0624769Y2 (en) | Test piece for chloride concentration measurement | |
JPH01101453A (en) | Detection of material to be analyzed | |
DE3263853D1 (en) | Activated apoglucose oxidase, method of preparing it, its use in specific binding assay methods and reagent means and test kits containing it | |
DK145286B (en) | METHOD FOR APPLYING A MEASURED QUANTITY OF A WATER SOLUBLE COUNTER WATER DISPERSIBLE REAGENT TO A WATER SOLID MEDIUM AND RELATED PROCEDURE TO ANALYSIS OF A TEST | |
JPS63111464A (en) | Detection of nitrite ion | |
Orbán et al. | Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level | |
Bright | Purification and iodination of actin | |
ATE220713T1 (en) | METHOD FOR IDENTIFYING BACTERIA AND DETERMINING THEIR SENSITIVITY TO ANTIBIOTICS AND DEVICE AND MEASUREMENT SUPPORT THEREFOR |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGE 1/1, DRAWINGS, REPLACED BY A NEW PAGE 1/1 |
|
ENP | Entry into the national phase in: |
Ref country code: CA Ref document number: 2321100 Kind code of ref document: A Format of ref document f/p: F Ref document number: 2321100 Country of ref document: CA |
|
ENP | Entry into the national phase in: |
Ref country code: JP Ref document number: 2000 532558 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09641674 Country of ref document: US |
|
NENP | Non-entry into the national phase in: |
Ref country code: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 27771/99 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1999908309 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1999908309 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |