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WO2006000999A1 - Support pour jeux ordonnes d'echantillons biologiques ou chimiques presentant une meilleure qualite de tache - Google Patents

Support pour jeux ordonnes d'echantillons biologiques ou chimiques presentant une meilleure qualite de tache Download PDF

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Publication number
WO2006000999A1
WO2006000999A1 PCT/IE2005/000070 IE2005000070W WO2006000999A1 WO 2006000999 A1 WO2006000999 A1 WO 2006000999A1 IE 2005000070 W IE2005000070 W IE 2005000070W WO 2006000999 A1 WO2006000999 A1 WO 2006000999A1
Authority
WO
WIPO (PCT)
Prior art keywords
support
membrane
media
nucleic acid
nylon
Prior art date
Application number
PCT/IE2005/000070
Other languages
English (en)
Inventor
Gerasimos Mouzakitis
David Bartholomew O'leary
Thomas Cotter
Original Assignee
University College Cork - National University Of Ireland, Cork
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University College Cork - National University Of Ireland, Cork filed Critical University College Cork - National University Of Ireland, Cork
Publication of WO2006000999A1 publication Critical patent/WO2006000999A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00387—Applications using probes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00639—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium
    • B01J2219/00641—Making arrays on substantially continuous surfaces the compounds being trapped in or bound to a porous medium the porous medium being continuous, e.g. porous oxide substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00691—Automatic using robots
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5088—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above confining liquids at a location by surface tension, e.g. virtual wells on plates, wires

Definitions

  • the invention relates to solid-phase hybridisation assays incorporating nucleic acids, in particular to gene arrays used for analysis of differential gene expression.
  • gene arrays evolved from Southern blot analysis, in which a single DNA fragment is hybridised to a multitude of immobilised fragments on a solid support (usually a nylon membrane) (Maskos and Southern, Nucleic Acids Res., 20, 1679-1684, 1992). This classical procedure has been reversed in gene arrays, in which a multitude of DNA fragments (called target DNA) are hybridised to individual DNA fragments (called probe DNA) immobilised on the membrane. Using such methods it is possible to analyse the differential gene expression of thousands of individual genes between different tissues, such as healthy and tumour cells, treated and untreated cell culture, and cells at different developmental stages. 0070
  • the data collected from a gene array analysis enable the piecing together of cellular pathways, and identification of gene/protein function(s).
  • probe DNA fragments are applied to a nylon membrane using manual or automated methods, forming "DNA spots".
  • the target DNA is hybridised to the immobilised probe DNA and the membrane is washed to remove un-hybridised target DNA.
  • intensity of each spot i.e. the level of target DNA bound to each probe DNA spot
  • differential gene expression between the samples is analysed.
  • a support for supporting high-density biological and chemical arrays comprising a membrane which has a medium applied thereto to enhance spot quality and/or spot density when probe material is applied in spots to the membrane and a target material is hybridised to the probe material.
  • the membrane comprises a fibrous material which is impregnated with the medium.
  • the membrane is of a polymeric material.
  • the membrane may be of a polyamide material.
  • the membrane may be of a nylon material.
  • the medium may be any suitable substance that is more hydrophobic than the probe material.
  • n is an integer from 4 to 16.
  • n may, for example, be 8 or 10.
  • the medium is oil.
  • the oil may be a mineral oil.
  • the medium comprises a mixture of an alkane and an oil.
  • the alkane may be dodecane or decane and the oil may be a mineral oil
  • the medium may contain other chemical substances: such as additives, stabilisers, preservatives, and/or anti-foaming agents, etc. Such substances may improve handling and/or stability of the medium or media-impregnated supports.
  • the probe material may be a nucleic acid such as RNA or DNA.
  • the invention also provides a method of carrying out an assay using a support of the invention.
  • the assay may be a solid-phase hybridisation assay.
  • the present invention provides for methods for altering the surface characteristics of membranes, especially nylon membranes, to allow for improved quality during the application of nucleic acid solutions.
  • the invention provides for chemicals and methods for impregnation of such membranes using at least one media, methods for application and attachment of nucleic acids, and methods for removal of the impregnation media.
  • nucleic acid is simply applied to the dry membrane.
  • the nucleic acid spots (which are applied from an aqueous solution) are allowed to dry and the nucleic acid is bound to the nylon.
  • the membrane is impregnated with media prior to application of the nucleic acids.
  • This treatment alters the surface characteristics of the support resulting in uniform, circular nucleic acid spots.
  • the membranes are exposed to the media which permeates the structure of the membrane.
  • the nucleic acids are applied to the media-impregnated support, dried, and processed as normal for binding. Following binding of the nucleic acids to the support, hybridisation reactions can be performed.
  • the impregnation media has a higher hydrophobicity index than the nucleic acid aqueous solution. Therefore, when the aqueous nucleic acid solution is applied to the slightly hydrophobic, media-impregnated nylon membrane it forms a circular, well-defined drop; this is similar to a droplet of water on an oily film.
  • the media serves to contain each droplet and prevents diffusion due to capillary action.
  • the nucleic acid spots are allowed to permeate into the media-impregnated membrane, dried and processed for binding the nucleic acid to the nylon as normal. Hybridisation can then be performed as normal.
  • nucleic acid solution applied to the media-impregnated support is delivered using non-contact methods, including, but not limited to inkjet technology.
  • a person knowledgeable in the art can select a suitable non-contact method for delivery of the nucleic acid solution to the media-impregnated membrane.
  • the impregnation media may or may not be volatile and so may or may not be removed.
  • the impregnation media is composed of a multiplicity of components, at least one component may be removed before or after the application of nucleic acids to the membrane.
  • Fig. 1 shows nucleic acid spots applied to nylon membranes impregnated with different media. Un - untreated; DE - Decane treatment; DO - Dodecane treatment; DM - Dodecane/Mineral Oil (4:1 volume/volume);
  • Fig. 2 is a graph showing the relative size of nucleic acid spots applied to media-impregnated nylon membranes
  • Fig. 3 shows the time course of removal of Impregnation Media
  • Fig. 4 is a graph showing the spot area during time course of removal of Impregnation Media
  • Fig. 5 shows the removal of Impregnation Media by using high temperature
  • Fig. 6 is a graph showing the spot area during removal of Impregnation Media by using high temperature.
  • the invention provides methods for altering the surface characteristics of membranes, including chemicals and methods for media impregnation of membranes, methods for application and attachment of nucleic acids and methods for removal of impregnated media.
  • the invention provides methods of impregnation of membranes with a medium to alter its surface characteristics, particularly in the application of nucleic acids.
  • the impregnation media is a chemical or a multiplicity of chemicals with a hydrophobicity index higher than that of the nucleic acid aqueous solution that will be spotted on the membrane.
  • a hydrophobicity index higher than that of the nucleic acid aqueous solution that will be spotted on the membrane.
  • the constituents of the impregnation media may or may not be volatile.
  • Impregnation of the membrane with the media may be performed using a wide range ' of methods, including, but not limited to immersion or spraying. A person knowledgeable in the art would be able to decide upon a suitable method. Regardless of the impregnation method used, the nylon membrane surface is evenly and homogeneously covered in the media.
  • the membrane is brought into contact with the impregnation media. Following a given period of time to allow for saturation of the membrane matrix, any excess impregnation media is removed. This ensures that there is a homogeneous environment on the membrane for depositing the aqueous nucleic acid solutions.
  • nucleic acid solutions into spots on media-impregnated membranes can be performed using a wide-variety of methods. A person knowledgeable in the art will be able to select the most appropriate method.
  • nucleic acids on to media-impregnated nucleic acids is performed using 'non-contact' methods, including, but not limited to, manual application by micro-pipette and inkjet technology.
  • non-contact methods only the nucleic acid solution contacts the membrane allowing for a circular, well-defined nucleic acid spot.
  • nucleic acid solution using 'contact' methods such as the mechanical stamping with pins
  • a pin covered in a nucleic acid solution comes into contact with a nylon membrane.
  • the nucleic acid is transferred on to the membrane by capillary action.
  • Such contact methods are not ideal though for media-impregnated membranes as the pins deposit the nucleic acid over a large area.
  • the hydrophobic media prevents capillary action, leading to variable transfer of nucleic acids from the stamping pin.
  • a multiplicity of nucleic acid solutions is applied on each membrane.
  • nucleic acid solution is applied on the media-impregnated membrane, they are allowed to enter the membrane matrix and dried under controlled conditions.
  • a person knowledgeable in the art can select any suitable method for hybridisation of nucleic acids to the immobilised nucleic and methods for detection of the hybridisation.
  • the impregnation media may be partially or fully removed from the membranes following the application of nucleic acids.
  • the constituents of the impregnation media may or may not be volatile.
  • any of the constituents of the impregnation media are volatile, they may be removed following the application of the nucleic acids using a range of methods, including, but not limited to, controlled ventilation and/or vacuum. A person knowledgeable in the art can select the most appropriate method.
  • Some methods for cross-linking nucleic acids to a support involve subjecting the nylon to a vacuum and/or increased temperatures. Using such procedures it is possible to cross-link the nucleic acids on to the membrane while simultaneously removing the volatile constituents of the impregnation media.
  • GAPDH nucleic acid solution Using mouse genomic DNA, in conjunction with GAPDH forward and reverse primers (see below), PCR was carried out under the conditions outlined in Table 1.1.
  • GAPDH forward primer sequence 5' ACC ACA ATC CAT GCC ATC AC 3'
  • GAPDH reverse primer sequence 5' TCC ACC ACC CTG TTG CTG TA 3'
  • PCR cycling conditions PCR amplification was performed using initial melting incubation at 95°C for 4 minutes, followed by 30 cycles at 95 0 C for 30s, 58 0 C for 30s, 72 0 C for 30s. Following amplification, products were maintained at 4 0 C.
  • GAPDH PCR amplification products were purified by electrophoretic separation. GAPDH PCR amplification products (5 ⁇ l) were mixed with l ⁇ l of 6X loading dye (0.05% bromophenol blue, 0.05% xylene cyanol, 15% glycerol,) and then separated on a 1% (weight/volume) agarose gel. PCR amplification products were visualised using ethidium bromide staining together with UV illumination. The appropriate GAPDH PCR amplification product was excised from the gel and purified by G-25 columns (Amersham Pharmacia).
  • 6X loading dye 0.05% bromophenol blue, 0.05% xylene cyanol, 15% glycerol,
  • the purified GAPDH PCR amplification products were quantified using a Hybaid spectrophotometer and diluted to 100 ng/ ⁇ l using sterile water.
  • b) Preparation of GAPDH nucleic acids for spotting on nylon membranes Purified GAPDH nucleic acid were denatured by heating to 95 0 C for five minutes and immediately placed on ice for 2 minutes. Nucleic acids were diluted to a final concentration of 2.5 ng/ ⁇ l in Spotting Buffer (TE and 0.01% weight/volume bromophenol blue dye). This GAPDH solution was used in the examples below.
  • Hybridisation of spotted nylon with labelled probe The prehybridisation/hybridisation buffer (PerfectHyb, Sigma) was heated in an oven to 42 0 C.
  • the spotted nylons were wetted with a 2X SSC and then each nylon membrane was put in a separate hybridisation tube (Hybaid) with 10ml of the prehybridisation/hybridisation buffer and incubated in an oven (rolling) at 42 0 C for two hours.
  • the prehybridisation/hybridisation buffer was discarded and replaced with fresh prehybridisation/hybridisation buffer (10ml) along with the 50 ⁇ l of labelled probe. This was incubated overnight rolling in an oven at 42 0 C.
  • Example 1 Chemicals and Methods for Media Impregnation of Nylon Membranes The invention provides for chemicals and methods for media impregnation of nylon membranes.
  • nylon membranes are cut into approximately two centimetre square pieces. Each nylon membrane piece is treated by complete immersion in media impregnation for five minutes at room temperature. Following this incubation, each nylon membrane is lightly blotted between two pieces of Whatmann 3MM paper to remove excess impregnation media from the surface of the nylon membrane.
  • membranes were treated with one of the following types of media: decane (DE), dodecane (DO), or dodecane/mineral oil (DM) (4:1 mixture volume/volume). Untreated (Un) nylon membranes were used as baseline controls. Immediately after each treatment, 2 ⁇ l of GAPDH solution (containing bromophenol blue dye, see above) was applied to each membrane in quadruplicate (Fig. 1). Due to the presence of the dye, the area of each nucleic acid spot was easily measured by densitometry.
  • DE decane
  • DO dodecane
  • DM dodecane/mineral oil
  • the average area of the four spots was calculated and expressed as a percentage of the average spot size in the untreated membrane (Fig. 2). Under these conditions, all three treatments decreased nucleic acid area to between 50% to 55% of the area in the untreated membrane. Dodecane treatment (DO) resulted in the smallest spots, approximately 51.5% of the area of nucleic acids spots on untreated membrane. It is important to note that either a single or a multiplicity of chemicals can be utilised to alter the spotting characteristics of the nylon membrane.
  • Example 2 Methods for Application of Nucleic Acids onto Media-Impregnated Nylon Membranes.
  • the invention also provides methods for application of nucleic acids onto media- impregnated membranes.
  • DNA nucleic acids were applied to media-impregnated membranes using four different methods.
  • a radioactively labelled probe was produced, hybridised to the nucleic acids on each membrane and visualised (as described under "General Solutions and Methodologies" sections c, d, e).
  • Non-contact manual spotting involved spotting 2 ⁇ l of the purified GAPDH PCR (lOOng/ul) diluted in TE to a concentration of 2.5ng/ ⁇ l. The spotting was performed by using a Gilson PlO to take up 2ul of the sample DNA into the gilson tip and then expressing the drop so that it hung from the tip. Then holding the Gilson PlO at a 45° angle to the nylon membrane, gently touching the drop on the surface of the membrane. This is a non-contact method as only the sample comes in contact with the membrane.
  • Non-contact robotic spotting Non-contact robotic spotting was carried out commercially (GeSiM, Deutschen fur Silizium Microsysteme).
  • a non-contact piezoelectric system was used to apply two spot sizes; 0.0378 ⁇ l and 0.0756 ⁇ l.
  • Spots consisted of GAPDH PCR diluted in TE to a concentration of lOOng/ ⁇ l. Ten spots of each size were applied to untreated nylon membranes and nylon membranes treated with various immersion media.
  • the invention also provides methods for removal of the impregnation media following application of the nucleic acid spots. Removal of impregnation media may be desirable to ensure that it does not interfere with any subsequent treatment of the nylon membrane and/or the applied nucleic acids.
  • Example 3a Removal of Volatile Media by Incubation at Room-Temperature Nylon membranes were treated and spotted with a nucleic acid/dye solution at different times post-immersion. Removal of media was monitored by measuring the area of the nucleic acid/dye spots at each time point. Untreated nylon membranes were used as baseline controls.
  • Nylon membranes were treated with decane (DE), dodecane (DO), or dodecane/mineral oil (DM, 4:1 ratio, volume/volume) as described in Example 1, above. Untreated membranes were used as controls.
  • DE decane
  • DO dodecane
  • DM dodecane/mineral oil
  • the average area of the two spots was calculated and expressed as a percentage of the average spot size in the untreated membrane for that time point (Fig. 4). Note that past 0.5 hrs post-immersion, the area of nucleic acids spots in the decane-treated membrane increases to almost the area of the untreated membrane. These results are consistent with removal of volatile media from the membrane by evaporation. Similarly, past 8 hours the spot area in the dodecane-treated membranes increases to untreated levels.
  • Example 3b Removal of Volatile Media by Incubation at High Temperature
  • a common procedure in the preparation of nylon membranes for nucleic acid hybridisations is the covalent linking of the applied nucleic acids by incubation at high temperature. It is therefore possible to utilise high temperatures to covalent link the applied nucleic acids to the nylon membrane while simultaneously removing a volatile impregnation media.
  • this Example also demonstrates that partial removal of the impregnation media may be preferable before or after application of the nucleic acid solution.
  • an impregnation solution may be applied to the nylon membranes and at least one component removed prior to application of nucleic acids.
  • nucleic acids If the nucleic acid is RNA it can be total RNA or mRNA (poly-A enriched). It can be a single RNA species, but most preferably it refers to a plurality of RNA species.
  • the nucleic acid is DNA it can be: genomic DNA, or fragments of genomic DNA, cDNA, an EST, a plasmid vector containing heterologous DNA nucleic acids, or an oligonucleotide. Furthermore, the DNA nucleic can be produced by purification, by an enzymatic process, or by synthetic chemistry. The DNA nucleic acid can be a single species or a plurality of DNA nucleic acid species.
  • nucleic acid a single or a plurality of nucleic acid species is referred to.
  • Solid-Phase Hybridisation Assay A solid-phase hybridisation assay is defined as an assay in which one nucleic acid is immobilised onto a solid support and hybridised to a non-immobilised nucleic acid. This type of assay includes (but is not limited to): Southern blot analysis, Northern blot analysis, DNA or RNA dot blots, and gene arrays.
  • Membrane A sheet of filamentous or fibrous or porous material that can be used in a solid phase hybridisation assays. The membrane can be of a polymeric material such as a polyamide, especially nylon.
  • Media Media can be an individual or a multiplicity of chemicals.
  • the media may or may not be volatile.
  • the media may impregnate the membrane to suppress capillary action.
  • suitable media are the higher alkanes such as decane or dodecane.
  • Suitable oils such as mineral oil may also be used.
  • target nucleic acid is defined as the non-immobilised nucleic acids in a solid phase hybridisation assay.
  • the probe nucleic acids are DNA as defined above (see Nucleic acids).
  • probe nucleic acids are defined as the immobilised nucleic acids in a solid phase hybridisation assay.
  • the target nucleic acids are most preferably DNA (as defined above in Nucleic acids definition) but may also be RNA.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un support pour jeux ordonnés d'échantillons biologiques ou chimiques à haute densité. Ce support comprend une membrane sur laquelle un milieu est appliqué afin d'améliorer la qualité de tache et/ou la densité de tache lorsqu'une matière de sondage est appliquée sur les membranes sous forme de taches. La membrane peut comprendre une matière fibreuse qui est imprégnée du milieu.
PCT/IE2005/000070 2004-06-24 2005-06-21 Support pour jeux ordonnes d'echantillons biologiques ou chimiques presentant une meilleure qualite de tache WO2006000999A1 (fr)

Applications Claiming Priority (2)

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US58208204P 2004-06-24 2004-06-24
US60/582,082 2004-06-24

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3418083A (en) * 1965-05-20 1968-12-24 Boehringer & Soehne Gmbh Hydrophobic test strip for analyzing whole blood
US4548906A (en) * 1981-11-02 1985-10-22 Fuji Shashin Film Kabushiki Kaisha Integral multilayer analytical element for the analysis of ammonia or an ammonia forming substrate and a method for the detection thereof using the same
EP0523457A1 (fr) * 1991-07-02 1993-01-20 Hitachi Software Engineering Co., Ltd. Méthode pour faire transparent une membrane nylon
US6355419B1 (en) * 1998-04-27 2002-03-12 Hyseq, Inc. Preparation of pools of nucleic acids based on representation in a sample
US20030036084A1 (en) * 1997-10-09 2003-02-20 Brian Hauser Nucleic acid detection method employing oligonucleotide probes affixed to particles and related compositions
US20030054384A1 (en) * 2000-01-11 2003-03-20 Maxygen, Inc. Integrated systems and methods for diversity generation and screening

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3418083A (en) * 1965-05-20 1968-12-24 Boehringer & Soehne Gmbh Hydrophobic test strip for analyzing whole blood
US4548906A (en) * 1981-11-02 1985-10-22 Fuji Shashin Film Kabushiki Kaisha Integral multilayer analytical element for the analysis of ammonia or an ammonia forming substrate and a method for the detection thereof using the same
EP0523457A1 (fr) * 1991-07-02 1993-01-20 Hitachi Software Engineering Co., Ltd. Méthode pour faire transparent une membrane nylon
US20030036084A1 (en) * 1997-10-09 2003-02-20 Brian Hauser Nucleic acid detection method employing oligonucleotide probes affixed to particles and related compositions
US6355419B1 (en) * 1998-04-27 2002-03-12 Hyseq, Inc. Preparation of pools of nucleic acids based on representation in a sample
US20030054384A1 (en) * 2000-01-11 2003-03-20 Maxygen, Inc. Integrated systems and methods for diversity generation and screening

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