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US 6890723 B2 Abstract A method of fabricating an array of biopolymers provides a shield for biochemical reactions and biochemical reactants and is particularly useful for those reactions and reactants that are susceptible to reaction with a component of the ambient environment during the fabrication of the array. The method is applicable to the conventional fabrication and synthesis methods used to fabricate a biopolymer array, such as in situ synthesis of biopolymers on an array and the attachment of pre-synthesized biopolymers on to an array. The method comprises applying a non-miscible fluid (NMF) to the array surface where the biopolymers are being synthesized or attached. The NMF is inert and insoluble with the biochemical reactants and other ancillary materials in solution used in conventional synthesis or attachment of biopolymers. The NMF provides a shield between the ambient atmosphere and the biopolymer synthesis materials or the deprotected pre-synthesized biopolymer at the surface of the array during the synthesis or attachment processes. The NMF may be applied as droplets over each feature location on the surface or may be applied by flooding the surface of the array to fully cover the features. Biomonomer or biopolymer solutions are deposited into or through the NMF to the feature locations on the surface of the array where the synthesis or attachment reactions are to take place using conventional deposition equipment to eject the solutions into the NMF. The NMF provides a shield for activated biomonomers that are susceptible to reaction with a component in the ambient environment, such as moisture in the air. Moreover, the NMF provides a shield for pre-synthesized biopolymers that are susceptible to evaporation when deprotected for attachment to the array surface. The method provides a means by which the potential reactivity of the activated biomonomer or deprotected biopolymer with an ambient atmosphere component can be kept low. As a result, biopolymer arrays can be more accurately fabricated.
Claims(19) 1. A method of fabricating a biopolymer array from pre-synthesized biopolymers, wherein the array has a surface that is prepared for linking with the pre-synthesized biopolymers, and wherein the pre-synthesized biopolymers are in solution for linking to the prepared surface, the method comprising the steps of:
adding a non-miscible fluid (NMF) to the array surface, the NMF being inert, immiscible and insoluble in aqueous solution; and
depositing the biopolymer solution on the array surface and linking the biopolymer to the surface.
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8. A method of shielding biosynthesis reactions and sensitive biosynthesis reactants from the ambient environment comprising the steps of:
(a) applying a non-miscible fluid (NMF) to one or more sites where the biosynthesis reactions take place, the NMF being inert and insoluble with respect to the biosynthesis reactions and the biosynthesis reactants, the NMF covering the one or more sites; and
(b) depositing one or more of the sensitive biosynthesis reactants through the NMF on the one or more sites.
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16. A method of fabricating a biopolymer array from pre-synthesized biopolymers, wherein the array has a surface that is prepared for linking with the pre-synthesized biopolymers, and wherein the pre-synthesized biopolymers are in aqueous solution for linking to the prepared surface, the method comprising the steps of:
adding a non-miscible fluid (NMF) to the array surface, the NMF being inert, immiscible and insoluble in aqueous solution; and
depositing the biopolymer solution on the array surface and linking the biopolymer to the surface,
wherein the NMF covers the deposited biopolymer solution during linkage.
17. The method of
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Description This is a divisional of copending application Ser. No. 09/426,823 filed on Oct. 22, 1999. This invention relates to arrays of biopolymers used in diagnostics, screening, gene expression analysis, and other applications. In particular, the invention relates to a method of fabricating biopolymer arrays, such as DNA arrays. Polynucleotide arrays, such as DNA or RNA arrays, are known and are used, for example, as diagnostic or screening tools. Such arrays comprise a plurality of different polynucleotide probes arranged in a predetermined configuration on a substrate. The polynucleotides of the plurality differ by having a different nucleotide sequence. Different polynucleotide probes are located at different regions (also known as features or spots) on the substrate, wherein in each region, multiple copies of the same polynucleotide are usually present. The array is exposed to a sample of biological material to be evaluated, also known as the �target�. Upon exposure to the target sample, the array will exhibit a binding pattern, wherein complementary target polynucleotides will hybridize or bind to the array polynucleotide probes during an assay. This binding pattern can be observed, for example, by labeling all polynucleotide targets (for example, DNA) in the sample with a suitable label (such as a fluorescent compound), and accurately observing the fluorescence pattern on the array. Assuming that the different sequence polynucleotide probes were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the target sample. Biopolymer arrays can be fabricated using either methods of deposition of intact biopolymer species or using in situ synthesis methods. The deposition methods basically involve depositing intact biopolymers at predetermined locations on a substrate that are suitably activated such that the intact biopolymers can link thereto. The intact species of biopolymers, each having different monomer sequences, may be deposited at different regions of the substrate to yield the completed array having a predetermined configuration. Typical procedures known in the art for deposition of intact polynucleotide species, particularly DNA such as whole oligomers or cDNA, are to load a small volume of DNA in solution in one or more drop dispensers such as the tip of a pin or in an open capillary and, touch the pin or capillary to the surface of the substrate. Such a procedure is described in U.S. Pat. No. 5,807,522. When the fluid touches the substrate surface, some of the fluid is transferred from the pin or capillary to the substrate location. The pin or capillary must be washed prior to picking up the next type of DNA for spotting onto the array. This process is repeated for the plurality of different polynucleotides and, eventually, the desired array having a predetermined configuration is formed. Alternatively, the DNA can be loaded into a drop dispenser in the form of an inkjet head and fired onto the substrate. Such a technique has been described, for example, in PCT publications WO 95/25116 and WO 98/41531, and elsewhere. The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA) using phosphoramidite or other chemistry. Such in situ synthesis methods can be basically regarded as iterative steps of depositing droplets of: (a) a protected monomer onto predetermined locations on a substrate to link with either a suitably activated substrate surface (or with a previously deposited deprotected monomer); (b) deprotecting the deposited monomer so that it can now react with a subsequently deposited protected monomer; and (c) depositing another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one cycle so that the different regions of the completed array will carry the plurality of different biopolymer sequences as desired in the completed array. In situ synthesis methods may require one or more intermediate further steps in each iteration, such as oxidation and washing steps, as are well known in the art. In order for an assay to yield accurate results, it is important that the different biopolymer features actually be present on the array, that they are put down accurately in the desired or predetermined pattern, that the biopolymers are of the correct size, and that each different feature be uniformly populated with the respective biopolymer. In polynucleotide arrays, the conventional in situ synthesis methods use phosphoramidite nucleoside monomers. In order for the phosphoramidite group to link to a hydroxyl of a previously deposited deprotected polynucleotide monomer, it must first be activated usually by using a weak acid, such as tetrazole. However, an activated phosphoramidite is highly reactive with moisture in the air. Therefore, unless some precaution is taken, the activated phosphoramidite can be used up before the desired reaction is complete. As a result there is a reduction in the deposited phosphoramidite monomer available for forming the complete polynucleotide. This problem is present even when the synthesis is performed in a nitrogen chamber. Further, the size (volume) of the synthesis droplet on the substrate surface could be very small, such as a few pico- or nano-liters, such that the ratio of surface to volume is very high. A high surface to volume ratio favors the diffusion of moisture into the droplets. Initially, the moisture from the air tends to be adsorbed at the surface of the synthesis droplet. Therefore, the phosphoramidite concentration at the surface of the droplet will tend to be lowest. Consequently, the concentration of a completed probe polynucleotide at a feature on the array tends to decrease from the center of a feature toward its perimeter. Variations in completed probe concentration within a feature result in a decrease in the concentration of target sample that consequently hybridizes to the respective polynucleotide probe. Therefore, the total signal that should be available from the hybridized target is diminished at the particular feature location during optical evaluation of the array. Further, it should be noted that the water vapor concentration in the ambient atmosphere might vary. Therefore, the signal from the hybridized target may also vary from array to array, leading to inconsistency in absolute signal generated from different arrays of a batch when the same concentration of a target is encountered. The foregoing problems exist particularly where the phosphoramidite is mixed with the activator and the mixture is deposited as a droplet on the substrate, and even where the activator is deposited onto a previously deposited droplet containing the phosphoramidite, both as such are described in PCT publication WO 98-41531. In either case, ambient moisture presents a problem. Furthermore, when one droplet is deposited on the other, there is no guarantee of efficient mixing such that the activated phosphoramidite will be evenly present at the substrate surface. Thus, it would be advantageous to have a means of fabricating biopolymer arrays that lessens the likelihood of deleterious environmental influences on the accuracy of the fabrication. In particular, it would be desirable, in the fabrication of arrays of biopolymers using biomonomers with a linking group that must be activated (such as a phosphoramidite), to provide a means by which the potential reactivity of the activated biomonomer with an ambient atmosphere component (such as water vapor in air) can be kept low. The present invention provides a method of fabricating an array of biopolymers on a substrate. The method is useful for shielding biosynthesis reactions and the bio-reactants from the ambient environment. Moreover, the method is useful for shielding pre-synthesized biopolymers during their attachment to an array substrate. In particular, the method is useful for shielding biosynthesis reactions and reaction components during the synthesis process or linking process that are susceptible to reaction with a component of the ambient environment, for example moisture in the air. The method of the invention is applicable to the conventional fabrication and synthesis methods used to fabricate a biopolymer array. In one aspect of the method, the array is fabricated using conventional in situ techniques. A first biomonomer is deposited onto a substrate for linking to a surface of the substrate in an array pattern of features by conventional methods. The linked biomonomer is deprotected using conventional methods, such that the biomonomer can react with subsequent biomonomers that are added to grow the biopolymer chain. The subsequent biomonomer may require activation before attachment to the growing chain. The biomonomer and a suitable activation reagent are deposited on the array for attachment to the deprotected surface-linked biomonomer. The biomonomers are deposited using conventional deposition equipment, such as computer controlled inkjet systems or piezoelectric deposition systems that are well known in the art. The method of the invention comprises applying a non-miscible fluid (NMF) to the array surface where the biopolymers are being synthesized. The NMF is inert and insoluble with the ancillary materials, reagents and biomonomers used in the synthesis of the biopolymers. In accordance with the invention, the NMF provides a shield between the ambient atmosphere and the biopolymer synthesis materials at the surface of the array during the synthesis process. The shield will delay the diffusion of ambient conditions into the synthesis areas. The subsequent biomonomers are deposited on the array until a desired biopolymer sequence is synthesized at each feature. The NMF may be applied as droplets over each feature location or may be applied by flooding the surface of the array to fully cover the features and the growing biopolymer sequences. The deposition system used to deposit biomonomers in solution on the array has a head with multiple pulsejets, each of which can dispense fluid droplets onto the substrate. Each such jet includes a chamber with an orifice, and an ejector which, when activated, causes a droplet to be ejected from the orifice. According to the invention, the inkjets of the deposition system are either immersed into the NMF to fire droplets of the activated biomonomer through the NMF to the biopolymer synthesis sites, or not immersed, but positioned above the level of the NMF to fire the activated biomonomer droplets into the NMF to the biopolymer synthesis sites on the array. In the preferred embodiment, the density of the NMF is different from the anhydrous solution of solvent, biomonomers and activation reagents. In a preferred embodiment, the method of fabricating further comprise the step of deactivating any unreacted activation reagent after the activated biomonomer is added to the linked biomonomer on the array. An ancillary material that stops the action of the activation reagent (i.e., deactivation reagent) may be added to the array, preferably by flooding the array surface. The deactivation reagent solution has a density that is different from the density of the NMF to facilitate the deactivation reagent reaching the unreacted activation reagent at biopolymer synthesis sites through the NMF shield. The preferred embodiment further comprises the step of removing all the ancillary materials and unreacted biomonomer from the array surface so that the growing biopolymer chain can undergo other chemistry. The foregoing steps are repeated, with a biomonomer deposited and linked to a previously deposited and linked biomonomer on the substrate. The growing biopolymer chain acts as a substrate bound moiety for each cycle, until all of the biomonomers have been added to the biopolymer array. In the fabrication of a typical array with multiple features, all of the foregoing steps are repeated at each of multiple different regions on the same substrate, where it is desired to form the biopolymer features. The biopolymers arrays that may be fabricated according to the invention include DNA, RNA, proteins, etc. arrays, for example. Where the array is a polynucleotide or oligonucleotide array (for example, DNA), the biomonomer is a nucleoside monomeric unit. The activated biomonomer is typically a phosphoramidite according to conventional oligonucleotide synthesis. Activated phosphoramidites are well known to be highly reactive with moisture. Without the method of the invention, an activated phosphoramidite will react with water vapor in ambient atmosphere and be depleted before a sufficient amount of the phosphoramidite has reacted with the growing polynucleotide chains of the array, even in a nitrogen chamber. In another aspect of the invention, a method of fabricating biopolymer arrays from pre-synthesized biopolymers is provided. The pre-synthesized biopolymer is deprotected before it is linked to the array surface. The deprotected pre-synthesized biopolymer is soluble in aqueous buffer solution. The droplets of the deprotected pre-synthesized biopolymer solution that are deposited for linking to an array substrate are very small and have the tendency to evaporate quickly in the ambient environment. The method of fabricating according to this embodiment comprises enclosing the droplets of the deprotected pre-synthesized biopolymer solution in the NMF for deposition. The NMF is inert, immiscible and insoluble in aqueous solution. Therefore, the NMF will surround the droplets and delay the diffusion of the aqueous solution out of the droplet such that the concentration of the deprotected pre-synthesized biopolymer will remain relatively constant while it links to the surface of the array substrate at each feature. In still another aspect of the invention, a method of shielding biosynthesis reactions and biosynthesis reactants from the ambient environment is provided. The method of shielding comprises applying the NMF to one or more sites where the biosynthesis reactions take place. The NMF is inert and insoluble with respect to the biosynthesis reactions and the biosynthesis reactants. The NMF is applied to cover the biosynthesis site(s). The method of shielding further comprises depositing one or more of the sensitive biosynthesis reactants through the NMF on the biosynthesis site(s). In still another aspect of the invention, a shield that protects sensitive biosynthesis reactions and biosynthesis reactants from the ambient environment is provided. The shield comprises a non-miscible fluid (NMF) applied to cover the biosynthesis reactions and reactants. The NMF is inert and insoluble with respect to the biosynthesis reactions and the biosynthesis reactants. The present methods and apparatus provide any one or more of a number of useful benefits. For example, in the fabrication of arrays of biopolymers using biomonomers with a linking group that must be activated, the present invention provides a means by which the potential reactivity of the activated biomonomer with an ambient atmosphere component can be kept low. Further, in the fabrication of arrays of biopolymers using pre-synthesized biopolymers that are water-soluble when deprotected for linking to the surface of the substrate, the present invention provides a means by which the potential reactivity of the deprotected biopolymer in solution with an ambient atmosphere can be kept low. The various features and advantages of the present invention may be more readily understood with reference to the following detailed description taken in conjunction with the accompanying drawings, where like reference numerals designate like structural elements, and in which: Definitions The following terms are intended to have the following general meanings as they are used herein: Polynucleotide�a compound or composition that is a polymeric nucleotide or nucleic acid polymer. The polynucleotide may be a natural compound or a synthetic compound. In the context of an assay, the polynucleotide can have from about 20 to 5,000,000 or more nucleotides. The larger polynucleotides are generally found in the natural state. In an isolated state the polynucleotide can have a bout 30 to 50,000 or more nucleotides, usually about 100 to 20,000 nucleotides, more frequently 500 to 10,000 nucleotides. Thus, it is obvious that isolation of a polynucleotide from the natural state often results in fragmentation. The polynucleotides include nucleic acids, and fragments thereof, from any source in purified or unpurified form including DNA, double-stranded or single stranded (dsDNA and ssDNA), and RNA, including t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA/RNA hybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomes of biological materials such as microorganisms, e.g. bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals, humans, and the like. The polynucleotide can be only a minor fraction of a complex mixture such as a biological sample. Also included are genes, such as hemoglobin gene for sickle-cell anemia, cystic fibrosis gene, oncogenes, cDNA, and the like. The polynucleotide can be obtained from various biological materials by procedures well known in the art. The polynucleotide, where appropriate, may be cleaved to obtain a fragment that contains a target nucleotide sequence, for example, by shearing or by treatment with a restriction endonuclease or other site specific chemical cleavage method. For purposes of this invention, the polynucleotide, or a cleaved fragment obtained from the polynucleotide, will usually be at least partially denatured or single stranded or treated to render it denatured or single stranded. Such treatments are well known in the art and include, for instance, heat or alkali treatment, or enzymatic digestion of one strand. For example, double stranded DNA (dsDNA) can be heated at 90-100� C. for a period of about 1 to 10 minutes to produce denatured material, while RNA produced via transcription from a dsDNA template is already single stranded. Oligonucleotide�a polynucleotide, usually single stranded, usually a synthetic polynucleotide but may be a naturally occurring polynucleotide. The oligonucleotide(s) are usually comprised of a sequence of at least 5 nucleotides, usually, 10 to 100 nucleotides, more usually, 20 to 50 nucleotides, preferably, 10 to 30 nucleotides, more preferably, 20 to 30 nucleotides, and desirably about 25 nucleotides in length. Various techniques can be employed for preparing an oligonucleotide. Such oligonucleotides can be obtained by biological synthesis or by chemical synthesis. For short sequences (up to about 100 nucleotides), chemical synthesis will frequently be more economical as compared to the biological synthesis. In addition to economy, chemical synthesis provides a convenient way of incorporating low molecular weight compounds and/or modified bases during specific synthesis steps. Furthermore, chemical synthesis is very flexible in the choice of length and region of target polynucleotides binding sequence. The oligonucleotide can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers. Chemical synthesis of DNA on a suitably modified glass or resin can result in DNA covalently attached to the surface. This may offer advantages in washing and sample handling. For longer sequences, standard replication methods employed in molecular biology can be used, such as the use of M13 for single stranded DNA as described in J. Messing (1983) Methods Enzymol. 101:20-78. Other methods of oligonucleotide synthesis include phosphotriester and phosphodiester methods (Narang, et al. (1979) Meth. Enzymol. 68:90) and synthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters 22:1859-1862) as well as phosphoramidite techniques (Caruthers, M. H., et al., �Methods in Enzymology, � Vol. 154, pp. 287-314 (1988) and others described in �Synthesis and Applications of DNA and RNA, � S. A. Narang, editor, Academic Press, New York, 1987, and the references contained therein. The chemical synthesis via a photolithographic method of spatially addressable arrays of oligonucleotides bound to glass surfaces is described by A. C. Pease, et al., Proc. Nat. Aca. Sci. USA (1994) 91:5022-5026. For the purposes of this invention, the terms �oligonucleotide� and �polynucleotide� are used interchangeably, unless otherwise noted. Oligonucleotide or polynucleotide probe�an oligonucleotide employed to bind to a portion of a polynucleotide, such as another oligonucleotide or a target nucleotide sequence. The design and preparation of the oligonucleotide probes are generally dependent upon the sensitivity and specificity required, the sequence of the target polynucleotide and, in certain cases, the biological significance of certain portions of the target polynucleotide sequence. Monomer�A member of the set of small molecules which can be joined together to form a polymer. The set of monomers includes but is not restricted to, for example, the set of common L-amino acids, the set of D-amino acids, the set of synthetic amino acids, the set of nucleotides and modified nucleotides, and the set of pentoses and hexoses. Other examples include a basic phosphodiesters, such as polyethers, and protein-nucleic acid (PNA) hybrids. As used herein, monomers refers to any member of a basis set for synthesis of a polymer. For example, dimers of the 20 naturally occurring L-amino acids form a basis set of 400 monomers for the synthesis of polypeptides. Different monomers may be used at successive steps in the synthesis of a polymer. Furthermore, each of the monomers may include protected members that are modified after synthesis. A monomer may also include modified monomers. Phosphoramidite�For the purposes of the invention, the term �phosphoramidite(s)� includes phosphite(s) and H-phosphonate(s). Formula I covers phosphoramidites, phosphites and H-phosphonates:
When x is equal to 1, R3 is an oxygen atom and R4 is an oxygen atom, the synthesis method is in this case the so-called phosphodiester method; when R4 is an oxygen atom carrying a protecting group, the synthesis method is in this case the so-called phosphotriester method. When x is equal to 1, R3 is a hydrogen atom and R4 is a negatively charged oxygen atom, the synthesis method is known as the H-phosphonate method. When x is equal to 0, R3 is an oxygen atom carrying a protecting group and R4 is either a halogen, the synthesis method is known as the phosphite method and, when R4 is a leaving group of the di-substituted amine type, the synthesis method is known as the phosphoramidite method. Phosphoramidites and nucleoside phosphoramidites are described in U.S. Pat. Nos. 5,902,878, 5,700,919, 4,415,732, PCT publication WO 98/41531 and the references cited therein, among others, all incorporated by reference. A �group� includes both substituted and unsubstituted forms. Nucleotide�the monomeric unit of nucleic acid polymers, i.e., DNA and RNA, that comprises a nitrogenous heterocyclic base, which is a derivative of either a purine or pyrimidine, a pentose sugar, and a phosphate (or phosphoric acid) and includes modified nucleotides. When the phosphate is removed, the monomeric unit that remains is a �nucleoside�. Thus a nucleotide is a 5′-phosphate of the corresponding nucleoside. When the nitrogenous base is removed from the nucleotide, the monomeric unit that remains is a �phosphodiester�. For the purposes of the invention, �nucleotide� includes its corresponding nucleoside and phosphodiester, and �oligonucleotide� includes its corresponding oligonucleoside and oligophosphodiester, unless indicated otherwise. Modified nucleotide�a unit in a nucleic acid polymer that contains a modified base, sugar and/or phosphate group. The modified nucleotide can be produced by a chemical modification of a nucleotide either as part of the nucleic acid polymer or prior to the incorporation of the modified nucleotide into the nucleic acid polymer. For example, the methods mentioned above for the synthesis of an oligonucleotide may be employed. In another approach a modified nucleotide can be produced by incorporating a modified nucleoside triphosphate into the polymer chain during an amplification reaction. Examples of modified nucleotides, by way of illustration and not limitation, include dideoxynucleotides, derivatives or analogs that are biotinylated, amine modified, alkylated, fluorophore-labeled, and the like and also include phosphorothioate, phosphite, ring atom modified derivatives, and so forth. Biomonomer�a single biological monomer unit, which can be linked with the same or other biomonomers to form a biopolymer. For example, a single amino acid or nucleotide with two linking groups, one or both of which may have removable protecting groups, is a biomonomer. A biomonomer fluid or solution or biopolymer fluid or solution refers to a liquid containing either a biomonomer or biopolymer, respectively, typically in a solution comprising an ancillary material. Biopolymer�a polymer found in biological systems comprising a plurality of biological monomeric units or biomonomers linked together, such as nucleic acids (including DNA, RNA, polynucleotides, oligonucleotides, oligonucleotide probes) sugars, proteins, antibodies, antigens, enzymes, coenzymes, ligands, receptors, hormones and labels, and genes that specify any of the above. Biopolymers include compounds composed of or containing amino acid or nucleotide analogs or non-nucleotide groups. This includes polynucleotides in which the conventional backbone has been replaced with a non-naturally occurring or synthetic backbone, and nucleic acids in which one or more of the conventional bases has been replaced with a synthetic base capable of participating in Watson-Crick type hydrogen bonding interactions. Substrate or surface�a porous or non-porous water insoluble material. The surface can have any one of a number of shapes, such as strip, plate, disk, rod, particle, including bead, and the like. The substrate can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like. Natural or synthetic assemblies such as liposomes, phospholipid vesicles, and cells can also be employed. Immobilization of oligonucleotides on a substrate or surface may be accomplished by well-known techniques, commonly available in the literature. See, for example, A. C. Pease, et al., Proc. Nat. Acad. Sci. USA, 91:5022-5026 (1994). Ancillary Materials�materials that are conventionally employed in the fabrication of biopolymer arrays. For example, ancillary materials include, but are not limited to buffers, salts, reagents, and solvents. For example, ancillary materials include activation, deactivation, deprotection, oxidizing, reducing and capping agents or reagents and anhydrous solvents used in biopolymer synthesis or in pre-synthesized biopolymer attachment to an array. The term �alkyl� refers to a monoradical branched or unbranched saturated hydrocarbon chain preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like, unless otherwise indicated. The term �alcohol� refers to an alkyl containing one or more hydroxyl groups. The term �alkyl alcohol� refers to a substituted alkyl having an alcohol group or a substituted alcohol having an alkyl group. The term �halogen� refers to fluoro, chloro, bromo and iodo groups. The term �haloalkylalcohol� or �halogenalkylalcohol� refers to an alkyl alcohol as defined above substituted by 1 to 4 halo groups as defined above, which may be the same or different, such as trifluoromethyl-, trichloroethyl- alcohol, and the like. It will also be appreciated that throughout the present application, words such as �upper�, �lower� and the like are used with reference to a particular orientation of the apparatus with respect to gravity, but it will be understood that other operating orientations of the apparatus or any of its components, with respect to gravity, are possible. Reference to a �droplet� being dispensed from a pulsejet herein, merely refers to a discrete small quantity of fluid (usually less than about 1000 pL) being dispensed upon a single pulse of the pulsejet (corresponding to a single activation of an ejector) and does not require any particular shape of this discrete quantity. When a �spot� is referred to, this may reference a dried spot on the substrate resulting from drying of a dispensed droplet, or a wet spot on the substrate resulting from a dispensed droplet which has not yet dried, depending upon the context. �Fluid� is used herein to reference a liquid. Use of the singular in reference to an item, includes the possibility that there may be multiple numbers of that item. A block diagram of the method 100 of fabricating an array of biopolymers on a substrate is illustrated in FIG. 1. The method 100 is useful for shielding the reactions and reactants in biopolymer synthesis, such as the growing biopolymer chain or a biomonomer to be added to the growing chain. In particular, the invention is useful for shielding reactions or reactants that are or become sensitive to a component of the ambient environment during the synthesis process. For example, the method is useful for shielding a biomonomer that, upon activation for attachment to the biopolymer, becomes susceptible to reaction with moisture in the air, or shielding a pre-synthesized biopolymer that, upon deprotection for attachment to an array substrate, becomes susceptible to evaporation. The method 100 of fabricating an array of biopolymers from biomonomers follows the conventional in situ synthesis chemistry for the synthesis of each type of biopolymer, such as oligonucleotides, proteins, etc. An array substrate is prepared so that a biomonomer can be linked to its surface using conventional methods. The biomonomer is deposited onto the surface of the substrate and is suitably linked thereto in an array pattern of features. According to conventional synthesis steps, the substrate-bound biomonomer is then chemically prepared to receive or link to another biomonomer. Different or the same biomonomers are subsequently added to the locations on the array features in an iterative process to synthesize the same or different biopolymers. The number of biopolymers and their sequence make-up, depends on the particular assay to be performed using the array. The biomonomer may be in solution comprising a solvent or other ancillary materials for deposition. For example, in the synthesis of oligonucleotides, typically the phosphoramidite is dissolved in an anhydrous solvent for deposition. Conventional anhydrous solvents that are used in phosphoramidite deposition are acetonitrile, propylene carbonate, adiponitrile, etc. The biomonomers that are subsequently added are chemically prepared, or may be activated, to link to the array bound biomonomer. In the synthesis of oligonucleotides, a phosphoramidite is activated by reaction with a reagent, such as tetrazole, ethylthiotetrazole, dicyanoimidazole, or benzimidazolium triflate, for example. Phosphoramidites and nucleoside phosphoramidites are well known in the art and described in, for example, U.S. Pat. Nos. 5,902,878, 5,700,919, 4,415,732, PCT publication WO 98/41531 and the references cited therein, among others, all of which are incorporated herein by reference. Therefore, the subsequent biomonomer solution may also contain an activation reagent or activator. Other conventional ancillary materials may be present also. A long-standing problem in in situ biopolymer array synthesis is the effect of environmental conditions on the synthesis process. For example, if one of the reagents or biomonomers is susceptible to reaction with components of the environment, for example, moisture in the ambient, the moisture will react immediately with the reagent or biomonomer until total hydrolysis thereof. Therefore, biopolymer synthesis is performed typically in a nitrogen chamber that is essentially devoid of moisture. However, biopolymer synthesis involves working with very small quantities in solution, such as droplets of a few pico- or nano- liters of product in solution. The surface-to-volume ratio of these droplets is very high and susceptible to even very small quantities of moisture. The high surface-to-volume ratio essentially favors the impact of diffusion of moisture into the droplets. Typical nitrogen chambers do not adequately prevent such small quantities of moisture, for example, from interfering with biopolymer synthesis. Therefore, without additional precautions, the droplets of reactive solution will react with components in the ambient environment, even in a nitrogen chamber, and will be depleted before all of activated biomonomer is allowed to react and become a part of the biopolymer. In accordance with the invention, the method 100 comprises applying 101 a non-miscible fluid (NMF) to the array surface where the biopolymers are being synthesized. The NMF completely covers the synthesis area(s) and provides an environmental shield for the synthesis reactants and reactions. According to the invention, the NMF is inert and insoluble with respect to the biomonomers, biopolymers, reagents and other ancillary materials used in the synthesis of the biopolymers. For oligonucleotide synthesis, the NMF is inert and insoluble with respect to the phosphoramidite biomonomer, its activator reagent and the anhydrous solvent, for example. For the purposes of the invention, the term �non-miscible� has its conventional definition. The NMF does not mix with the biomonomer, reagents, anhydrous solvents and other ancillary materials used in the synthesis processes. The term �inert� has its conventional definition. The NMF does not chemically react with the biomonomer, reagents, anhydrous solvent or other ancillary materials. Moreover, the term �insoluble� has its conventional definition. The solubility of the NMF in the solution of biomonomer and activator reagent in the anhydrous solvent is less than about 10 mg per milliliter. In addition, the NMF should have a low affinity for the environmental component(s) in the ambient atmosphere that the NMF is intended to shield against. For example, the NMF should have a low affinity for water, such that moisture vapor does not penetrate the NMF to reach a biomonomer that is sensitive to moisture. The solubility of water in the NMF is below 0.2 g of water per kilogram of NMF at 20� C., and preferably below 0.13 g of water per kilogram of NMF at 20� C., and depends on the particular NMF. Some NMF that are particularly useful for the invention are listed in Table 1. The list in Table 1 is illustrative only. As long as the NMF has the characteristics described above, it is within the scope of the invention. In the case of oligonucleotide synthesis, preferably heptane provides the characteristics necessary for shielding phosphoramidite synthesis conditions and materials, from the ambient, but any other fluid with similar properties to heptane could be used. However, because of the melting temperature of heptadecane, the temperature must be kept above 20� C. when heptadecane is used as the NMF. The method 100 of the invention further comprises the step of depositing 103 the biomonomer solution, which is suitable activated for attachment to the surface of the array, or to a surface-bound biomonomer on the array, with conventional deposition equipment, such as computer controlled inkjet systems or piezoelectric deposition systems that are well known in the art. The deposition system used to deposit biomonomers in solution on the array has a head with multiple pulsejets, each of which can dispense fluid droplets onto the substrate. Each such jet includes a chamber with an orifice, and an ejector which, when activated, causes a droplet to be ejected from the orifice. There is typically one pulsejet for each different biomonomer to be deposited. The ejected droplet has sufficient speed to penetrate the NMF and reach the surface of the array where the biopolymer is being synthesized. According to one embodiment of the method 100, the step of depositing 103 comprises the step of immersing the pulsejets into the NMF before the step of ejecting droplets of the biomonomer solution. As mentioned above, there is one pulsejet 26 for each different type of biomonomer being deposited. For oligonucleotide synthesis, there are four pulsejets, for example, one for each nucleotide base. In practice, all pulsejets 26 may be immersed into the NMF in the step of immersing. However, not all of the pulsejets 26 will actually fire and eject a biomonomer solution 28 at each step of ejecting. Whether and which pulsejet 26 actually fires a biomonomer solution 28 depends on the desired sequence of biomonomers at each synthesis site 22 of the array 20 and is controlled by a computer. In each of the above embodiment, preferably the density of the NMF is different from the density of the biopolymer, biomonomer and ancillary materials. More preferably, the density of the NMF in each of the embodiments illustrated in FIGS. 2-4, 6 a and 6 b is lower than the density of the biopolymer, biomonomer and ancillary materials so that the NMF better covers or shields the synthesis site 22 while the biosynthesis reactions occur on the surface 23. In the embodiment of the method 100′ illustrated in As noted above, in each of the embodiments described above, the density of the activated biomonomer solution 28 is different from the NMF by being either higher or lower than the NMF, so that the biomonomer solution 28 effectively travels through the NMF to the synthesis sites 22 on the array surface 23 where the droplet remains for the synthesis to take place. In addition to differences in density, the surface tension of the different fluids facilitates the movement of the biomonomer droplets 28, as well as the deposition system. The inkjet and piezoelectric deposition systems can fire the droplets 28 at a high speed through the NMF shield to reach the surface 23 of the array 20 without difficulty. For example, for oligonucleotide synthesis, the firing speed of the pulsejet 26 is about 10 m/s. Therefore, the affect of having an NMF with a different density from the biomonomer droplets 28 is primarily to allow the NMF to surround and shield the activated biomonomers at the synthesis sites 22. The anhydrous NMF separates the synthesis sites 22 and all of the reactants from the atmosphere and shields them temporary from detrimental environmental conditions, such as moisture. The NMF delays the hydrolysis of reagents with moisture and allows the coupling reaction between biomonomers to be completed before moisture reaches the synthesis sites 22. Referring back to Upon deactivation, the method 100 preferably further comprises the step of removing 107 all the reagents and solutions from the array surface 23 so that the growing biopolymer chains can undergo further synthesis chemistry. Typically, one or more of the foregoing steps are repeated (step 109) in an iterative fashion, with a subsequent biomonomer 28 being deposited and linked to a previously deposited and linked biomonomer on the substrate 20. The growing biopolymer chain acts as a substrate bound moiety for each cycle, until all of the biomonomers have been added to the biopolymer array. In the fabrication of a typical array with multiple features, all of the foregoing steps are repeated at each of multiple different regions 22 on the same substrate 20, where it is desired to form the biopolymer features. The biopolymers that may be synthesized according to the invention include DNA, RNA, proteins, etc. Where the array is a polynucleotide or oligonucleotide array, the biomonomers are nucleoside monomeric units. Likewise, where the array is a protein array, the biomonomers are amino acids. Protein arrays can be arrays of antibodies, antigens, ligands and receptors also, for example. Proteins are synthesized according to conventional methods, such as those described in U.S. Pat. Nos. 4,591,570; 5,143,854; and 5,252,743 and the following articles: Ekins, R., et al., �Development of microspot multi-analyte ratiometric immunoassay using dual fluorescent-labeled antibodies� Analytica Chimica Acta, (1989), 227:73-96; and Ekins, RP and FW Chu. �Multianalyte microspot immunoassay�microanalytical �compact disc� of the future� (1991), all of which are incorporated by reference. In the case of oligonucleotide arrays, conventional in situ synthesis chemistry is used. The phosphoramidite monomer unit is activated typically with tetrazole, for example, before the coupling reaction. The activated phosphoramidite is moisture sensitive and will react immediately with moisture in the ambient to hydrolyze the phosphoramidite. Without the method 100, 100′ of the invention, the activated phosphoramidite will react with water vapor in ambient atmosphere, even in small amounts present in a nitrogen chamber, and be depleted before a sufficient amount of the activated phosphoramidite has reacted with the growing oligonucleotide chains of the array. In still another embodiment, a method 200 is provided for fabricating arrays of biopolymers, wherein the biopolymers are pre-synthesized and then linked to the surface 23 of an array 20 in a desired array pattern of features. Such arrays 20 of pre-synthesized biopolymers include, for example cDNA arrays, DNA arrays or RNA arrays. In accordance with the method 200, the surface 23 of the array substrate 20 is prepared to receive or link to the pre-synthesized biopolymer using conventional well-known methods. For example, when the biopolymer is cDNA, the array substrate surface 23 is coated with an aqueous solution of polylysine. Moreover, the protecting group on the pre-synthesized biopolymer is removed (deprotection) using conventional methods, so that the biopolymer can link to the prepared surface 23. The deprotection step renders the pre-synthesized biopolymer water-soluble. Therefore, the deprotected pre-synthesized biopolymer is dissolved in aqueous solution for deposition onto the array 20. For example, when the pre-synthesized biopolymer is DNA or RNA, the deprotected pre-synthesized DNA or RNA is water-soluble and is typically dissolved in an aqueous buffer solution for deposition. However, the aqueous solution of pre-synthesized biopolymer is susceptible to evaporation, especially in small quantities, for example, when it is deposited onto the surface of the array for linking. Unless some precaution is taken, the aqueous solution may prematurely evaporate before the linking reaction occurs, or before the linking reaction is completed. As a result, some of the pre-synthesized biopolymer will not link to the surface 23. Therefore, the evaporation of the aqueous solution on the surface will vary the concentration, and resulting uniformity, of deposited pre-synthesized biopolymers at the respective feature locations 22. In accordance with the method 200, the feature locations or sites 22 of the array 20 are then covered with the NMF (step 201). The NMF may be flooded over the surface 23, as in Droplets of the aqueous solution of deprotected pre-synthesized biopolymer 28′ are deposited (step 203) on the array 20, for example, by the pulsejet 26, as mentioned above for method 100, 100′, or with a pin or capillary, as is well known in the art. In the preferred embodiment, the pulsejet heads 26 deliver the different biopolymers 28′ to their desired feature locations 22 on the array 20. As described above for method 100, 100′, the pulsejet 26 fires the droplets of biopolymer 28′ into the NMF to the sites 22 or feature locations on the surface 23 of the array 20, where the particular biopolymer 28′ sequence is to be linked. Any of the deposition methods illustrated in In accordance with the invention, the NMF that is applied to the array 20 is insoluble in aqueous solutions, such as in water and in buffer solution, such as the buffer solution used for the deprotected pre-synthesized DNA or RNA. The NMF effectively shields each droplet 28′ and impedes or delays the diffusion and subsequent evaporation of the aqueous solution, so that the linkage reaction can be completed. As a result, the deposited pre-synthesized biopolymers 28′ have uniform concentration throughout each feature location 22. The NMF is then removed (step 204) from the array 20 and the array 20 is washed with water to remove any unbound biopolymers and ancillary materials, preferably about 5 to 10 minutes after all of the pre- synthesized biopolymers have been deposited. If the array surface 23 was treated with polylysine to prepare the surface for linkage, an additional step of capping unreacted polylysine surface sites 22 must be performed according to conventional methods to prevent further binding to biopolymers, such as target sequences during an assay, which could compromise assay results. Whether an additional step to cap or deactivate is needed generally depends on the biopolymers that are being linked and the type of linkage chemistry chosen. The additional steps that may be necessary are well known in the art. The methods 100 and 200 of the invention provide a robust tool for solving a longstanding problem in the art of making biopolymer arrays with accuracy. The biopolymer arrays are used in assays of target biological materials. In order for an assay to yield accurate results, it is important that the different biopolymer features actually be present on the array, that they are put down accurately in the desired or predetermined pattern and biomonomer sequence, that the biopolymers are of the correct size, and that each different feature be uniformly populated with the respective biopolymer. The present method 100, 200 provides means to protect the sensitive reaction environment during biopolymer array fabrication from external environmental influences that might compromise the fabrication of the desired biopolymer array. Moreover, the present invention provides a means by which the potential reactivity of an activated biomonomer or of a deprotected pre-synthesized biopolymer with an ambient atmosphere component can be kept low. As a result, the biopolymer array fabricated in accordance with the invention will more likely have uniformly populated biopolymers of the correct sequence and size at each array feature. While biopolymer arrays are described above, the present invention contemplates that these particular moieties can readily be replaced with other moieties (such as other chemical or biochemical moieties, for example various small molecules), where the activated component is more reactive with a component of the ambient atmosphere than is the unactivated component. Thus, wherever a reference is made to biopolymers, this can be replaced with a reference to any such moieties. The present invention is essentially a method of shielding sensitive biosynthesis reactions and reactants from one or more components of the ambient environment. The NMF is essentially a shield that is applied over the biosynthesis reactions to separate the bioreactants from the ambient environment while the biosynthesis reactions take place. Thus there has been described a new method 100, 100′, 200 of fabricating biopolymer arrays that provide a shield for sensitive biopolymer fabrication on the array from environmental conditions which may have detrimental effects on the accuracy of the fabrication results. It should be understood that the above-described embodiments are merely illustrative of the some of the many specific embodiments that represent the principles of the present invention. Clearly, numerous other arrangements can be readily devised by those skilled in the art without departing from the scope of the present invention. Patent Citations
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COMMERCIAL DESCRIPTION It offers a finely wooded nose hinting at hazelnut, and is therefore distinct from traditional British scotches. It has a more rounded taste letting a discreetly full-bodied bitterness come to the fore, and will naturally seduce you. Ingredients: Water, pale ale malt, caramel malt, aromatic malt, sugar, yeast, Kent hop, hallertau hop.
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Student Employment Job Fair
Wednesday, March 19, 2014 from 11:00am to 1:30pm
Strand Union Building, Ballroom B
Strand Union Building, Ballroom B
Meet with employers and apply for a variety of positions on and off campus. These are local employers offering part-time positions, internships, and summer/seasonal work geared around a student's schedule. Now is the time to... Get to Work!
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You are hereGeneral Discussion
General Discussion
Hillary Under Fire
By Dave Lindorff
So Hillary Clinton, her campaign sagging as it becomes clearer and clearer that she's not going to get the Democratic nomination unless she can destroy her opponent, insists she would have quit Barack Obama's Trinity United Church of Christ in Chicago after hearing the allegedly "hateful" words of its pastor, Jeremiah Wright.
News of Rapid Glacial Melting Raises a Big Question for Presidential Candidates
By Dave Lindorff
Okay. Enough about race.
We've got a bigger problem here than how to get along with each other, as important as that may be, and that's how to make sure that any of us--or our children and grandchildren--are around in another hundred years.
4000 US Dead in Iraq: Maybe What We Need is a National Spittoon in D.C.
By Dave Lindorff
Well, the toll of wasted American lives in Iraq has hit 4000. But hey, who's counting?
Certainly not the folks in the White House and the Pentagon, and certainly not John McCain, the prospective Republican nominee for president, who thinks the war is going just dandy.
Truth On CNN! Pundits Defend Obama! Anderson Cooper Is Perplexed! (As Usual)
In this seemingly endless presidential campaign, parsed through the drama and hyperbole of media, it is a rare and welcome occasion when reason and honesty eclipse tantrums and untruths. It is particularly rare when pundits buck the popular trend and speak truth to corporate power. Such an occasion, or mitzvah as some might say, occurred on March 20th (2008) on CNN, when veteran political analyst David Gergen, renowned reporter and author Carl Bernstein, and CNN commentator Roland Martin, shredded the race card and taught a lesson in truth to neophyte Anderson Cooper.
For the past several days, Anderson Cooper, an unseasoned political observer, has been pushing the race card as far as possible on all things Barack Obama. It's not that Cooper is any worse at assailing Obama than his counterparts at Fox, MSNBC and conservative talk radio. It's just that Cooper as a political anchor, is sorely unskilled. He's fine traversing the planet, reporting from rugged terrain. He loves pitting himself against nature -- be it a hurricane, tornado, earthquake or drought. He's terrific in stories on wildlife, and daring in the throes of war. But a savvy political anchor he is not. Nor is he a host much concerned with the ramifications of his remarks. Sadly, CNN in this election cycle, is nearly as treacherous as Fox. But on one brilliant evening in March, three media pundits put Cooper in his place by refusing to derail Barack Obama.
Here's how it happened:
Vermont Vets Risk All for the Truth
by Dan DeWalt
"I can't go back in time and take back what I've done… At one point I was a monster, and I created hate and destruction amongst many people. I am sorry for doing so and I will never turn back into the monster I
Five Years of a Disastrous War and the Bills are Coming Due
By Dave Lindorff
It's appropriate that on this week of the fifth anniversary of the criminal US invasion of Iraq, we are also seeing several other things: the death toll of American troops in that doomed adventure is rising past 4000, the economy is sliding into a recession which could be deep and long, and the financial markets are teetering on the edge of a possibly historic collapse.
The conjunction of all of these dire things is no coincidence.
Corporate Media Ignored Winter Soldier. Senators Biden & Kerry - Will YOU??
Corporate owned networks and cable TV are private for profit enterprises driven by MONEY -- not by truth. Network and cable "news" is private for profit infotainment driven by MONEY -- not by truth. Delivering truth and educating viewers are not the goals of corporate media. Increasing viewership, attracting advertisers, and cronyism are. Even when reporting the invasion of Iraq, corporate media mitigates or inflames the story to advance its selfish goals.
Censorship and the Anemic State of Political Discourse in America
By Dave Lindorff
When I lived in China in the early 1990s, there were things that you could not discuss. One was Tibet. Another was Taiwan, "referred to in my daughter's public elementary school in Shanghai as "China's largest island." Another was the 1989 massacre of students and workers in Beijing. I used to be grateful at the time that I was an American and that back home, we could talk about anything.
Except that in a way we can't. Not in public discourse, anyhow.
It's the "Oh Shit!" Moment on Iran
By Dave Lindorff
Every horror movie has that "Oh Shit!" moment, when the hero or heroes are huddled in some creepy hideout, and suddenly something happens that tells you that the monster is just around the corner, or just about to attack. In "Jurassic Park" it was the pulsing ripples in a cup of water, heralding the arrival of a T-Rex. In "Jaws" it was the deep base music, letting you know that a monstrous shark was about to attack.
The Presidential Assasination Attempt That Didn't Bark
An exhaustive review of more than 600,000 Iraqi documents that were captured after the 2003 U.S. invasion has found no evidence that Saddam Hussein's regime had any operational links with Osama bin Laden's al Qaida terrorist network.
Spitzer Bust Provides a Warning Regarding NSA Spying
By Dave Lindorff
I have no sympathy for New York Gov. Eliot Spitzer, the hot-shot prosecutor of call-girl operations who was hoist on his own petard, as it were. I mean, what a jerk! And aside from the hypocrisy, what a fine message he was sending to his three teenage daughters about the role of women.
Having said that, Spitzer's bust should give pause to those in Congress who are ready to hand President Bush a free pass to continue his six-year campaign of warrantless spying on Americans.
Is Spitzer Being Targeted By The Anti-Abortion Right?
Isn't it odd that just when New York Governor Elliott Spitzer has introduced the RHAPP Bill -- the Reproductive Health and Privacy Protection Act -- which guarantees women the right to choose or refuse birth control and abortion, he is simultaneously the subject of a highly publicized sex sting that threatens to destroy his career and possibly send him to prison?
America: Lying Politicians, Gullible Voters and the Long Slide Ahead
By Dave Lindorff
Well, fellow Americans, we are finally going to get our just rewards.
That "Almighty Dollar" you used to hear about is heading straight towards the scrap heap of junk currencies (think Ruble, Rupee, Lira, Escudo).
Iraq War To Cost $12B A Month In 2008
Iraq War To Cost $12B A Month In 2008
Book Projects War's Cost In 2017 To Be $1.7T To $2.7T
The flow of blood may be ebbing, but the flood of money into the Iraq war is steadily rising, new analyses show.
In 2008, its sixth year, the war will cost approximately $12 billion a month, triple the "burn" rate of its earliest years, Nobel Prize-winning economist Joseph E. Stiglitz and co-author Linda J. Bilmes report in a new book.
Racist KellyAnne Conway Sends Obama To THE BACK OF THE BUS
On last night's Larry King Live show (Friday, March 7th), Republican strategist and owner of The Polling Company, KellyAnne Conway, stated that Hillary Clinton and Barack Obama were
"arguing about whether she should let him SIT ON THE BACK OF THE BUS of her presidential ticket."
Even by CNN standards, with its ample chicanery and spin, Conway's remark was brutal. Larry King's nightly program, usually more schmooze than news, was plunged to a Limbaugh-like low. Gratefully, Democratic strategist and Obama supporter, Jamal Simmons, was on hand to challenge Conway. Taking the high road, Simmons said:
"Larry, we have to address that. We have to address that. This BACK OF THE BUS comment is really beyond the pale. I think Senator Clinton, whatever our disagreements are between the two candidates, Senator Clinton has never done or said anything that would imply that she would rather have Barack Obama sit in the back of her campaign bus."
A Manchurian Candidate in the White House?
By Dave Lindorff
With a viral campaign underway via email, right-wing radio, and on the street suggesting that Barack Obama is a black "Manchurian Candidate," secretly trained as a Muslim fanatic who will insinuate himself into the White House, thence to undermine all that we hold dear, perhaps it is time to look at the Manchurian Candidate we already have in the White House, who, together with his handler over in Blair House, has pretty much done all the damage already.
Welch Whitewash: We Still Don't Know What That Aug. 30 Nuke Incident Was About
By Dave Lindorff
A new report on the August 30 incident in which six nuclear-armed advanced cruise missiles were effectively "lost" for 36 hours, during which time they were, against all regulations, flown in launch position mounted on a pylon on the wing of a B-52H Stratofortress, from Minot AFB in North Dakota across the continental US to Barksdale AFB in Louisiana, has left unanswered some critical questions about the event.
McCain and the Lobbyist: Missing the Story of the Miss
By Dave Lindorff
All the attention in the breaking story about John McCain's 2000 relationship with a blonde young telecom lobbiest has been focussed on the question of whether or not they were "doing it."
As George Stephanopoulos claimed on ABC, the importance of the story depends upon whether McCain is shown to have had a "relationship" with the lobbiest, Vicki Iseman.
But really, who cares whether they were shacking up on the campaign trail? McCain, after all, already double-timed his starter wife and dumped her for a trophy wife, the statuesque and wealthy beer industry heiress Cindy Hensley, so it's not as though he is campaigning on a strong pro-family platform.
Portland ME Holds 1st Bi-Partisan Event on Constitutional Issues Resulting from Bush-Cheney Misadministration
Our Constitution In Crisis
What Does A Broken Democracy Look Like & How Do We Fix It?
Announcing a Public Forum With Maine's 1st Congressional District Candidates
PORTLAND, ME - February 18 - The future of the U.S. Constitution and the role of Congress will be discussed by the Candidates for Congress vying to replace Tom Allen representing Maine's First Congressional District. John Nichols, Washington correspondent for The Nation magazine, will set the stage at the forum by addressing the constitutional issues that have been created during the current Bush-Cheney Administration. The forum will be held on Tuesday evening, February 26, at 7 PM at First Parish Unitarian Universalist Church, 425 Congress Street in Portland. The public is invited and will have an opportunity to ask questions.
Bush/Cheney Adminstration Takes Us for Idiots
By Dave Lindorff
Anybody who thinks that the government is telling the truth about the plan to shoot down a dead spy satellite--that it's all about protecting us, and not about testing an anti-satellite weapon--has to be really stupid.
And stupid is what the Bush administration and the Pentagon apparently think we are.
They're claiming they need to shoot this thing down because it's got a tank with 1000 pounds of hydrazine, a fuel used for maneuvering satellites while in orbit which is said to be as toxic as chlorine if breathed.
Bush's Protect America Bill Bull
By Dave Lindorff,
President Bush has turned to the cheapest lies in an effort to protect himself from being exposed as a criminal in the ongoing campaign to have the National Security Agency spy at will on Americans.
Claiming—without a scintilla of evidence to back him up—that there are people planning a "much worse attack" than 9-11 on America, he says he must not only have free rein to unleash the NSA
Obama and Progressive Change
By Dave Lindorff
I don't want to overstate the case for Barack Obama, who has been fairly circumspect about his intentions if elected. While saying he is against the Iraq War, he has not acted very forcefully to help bring it to an end. And he certainly has not called for any downsizing of America's bloated military budget or any end to its imperialist foreign policy—absolutely essential if there is to be any progressive change of consequence in the US.
Cannon Beach OR City Council Passes Bush-Cheney Impeachment Resolution; Posthumously Honors Gainor Minott
Cannon Beach split on whether Bush and Cheney must go
Impeachment resolution passes 3-2 as neighbors work to comply with last wishes of resident Minott
By PAMELA ROBEL | The Daily Astorian
CANNON BEACH - In a split vote on Super Tuesday, during the regular meeting of the Cannon Beach City Council, a resolution was passed to impeach President George Bush and Vice President Dick Cheney.
The call for impeachment began with the last wishes of Cannon Beach resident Gainor Minott, who asked Rex and Diane Amos to pursue an impeachment of the president and the vice president. The Amoses collected 227 signatures from residents of Cannon Beach to present to the council in support of a call for impeachment. Minott died in September.
To Hell with the Chief!
By Dave Lindorff
What is all this nonsense about us electing a "commander in chief"?
Okay, I mean we all know it's President Bush's favorite title. He thrills to being a "war president," and loves strutting around in front of guys in uniform and getting saluted. But really, what is this all about?
The Constitution says that the President is the commander in chief of the army and the navy, and of the militia if it is called to national service. But that is really just for the sake of having a civilian at the top of the food chain.
In an unscheduled rally appearance at the University of California-Los Angeles' (UCLA) Pauley Pavillion, California's First Lady, Maria Shirver, surrounded by her cousin Caroline Kennedy, best friend Oprah Winfrey, and co-political spouse, Michelle Obama, publicly announced her endorsement of Barack Obama for President of the United States. With today's endorsement, Shriver now joins a long list of her Kennedy relatives who are endorsing Barack Obama.
Ralph Di Gia
December 13, 1914 – February 1, 2008
Ralph DiGia, World War II conscientious objector, lifelong pacifist and social justice activist, and staffer for 52 years at the War Resisters League (WRL), died February 1 in New York City. He was 93.
DiGia was "without pretensions, one who wore his radicalism in his life, not on his sleeve," said his long-time WRL colleague David McReynolds.
In addition to his decades at WRL, DiGia's activism took him through countless arrests and a stretch in federal prison, thousands of meetings and hundreds of demonstrations, hunger strikes, a bicycle ride across Europe, relief work in Bosnia, and not a few New York Mets baseball games.
80 Years of Activism
Born in the Bronx to a family of Italian immigrants in 1914, DiGia grew up on Manhattan's Upper West Side. A 1927 rally for Italian anarchists Nicola Sacco and Bartolomeo Vanzetti
This Campaign's a B-Movie with a B List of Characters
By Dave Lindorff
With the presidential race now effectively pared down to four candidates, thanks to the departure of John Edwards and the imminent departure of Rudy Giuliani, we're left with a really "B-grade" contest: a bomber (John McCain), a bummer (Hillary Clinton), a betrayer (Mitt Romney) and Obama (that's Barack with a B).
McCain, the bomber, wants to "bomb, bomb, bomb, bomb, bomb Iran," ensuring not just mayhem in Persia, but chaos, bloodshed and disaster across the Middle East and surely retaliation here at home for a generation to come. Here's a guy who made his name by getting himself shot down while committing war crimes against the people of a backward nation in Southeast Asia, who glommed onto Sen. Russ Feingold's clean government initiative after carting off wheelbarrows of cash from Charles Keating in the savings and loan scandal (McCain was one of the infamous Keating Five), and who claimed to be a renegade Republican and "straight talker" until he decided in 2004 to cling tightly to President Bush and become the mad Texan's biggest apologist during his second benighted term. And, after months of pandering to the Christian right and the neocon loonies, he now wants us to believe he's a maverick and "middle-of-the-roader."
El Diario/La Prensa Editorial: 935 Reasons for Impeachment
935 Reasons for Impeachment | El Diario/La Prensa
In the two years following the Sept. 11 attacks, President Bush and his right-hands—Vice President Dick Cheney, National Security Adviser Condoleezza Rice, Secretary of State Colin Powell, and Defense Secretary Donald Rumsfeld—made at least 935 false statements about the national security threat posed by Saddam Hussein.
Judge Orders Bush Administration to Explain Evidence Handling in CIA Videotapes Case
Judge Wants Answers on CIA Videotapes | ABCNews.com
Judge Orders Bush Administration to Explain Evidence Handling in CIA Videotapes Case
A federal judge said Thursday that CIA interrogation videotapes may have been relevant to his court case, and he gave the Bush administration three weeks to explain why they were destroyed in 2005 and say whether other evidence was destroyed.


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UNWTO saddened by the crash of Air Algerie flight UNWTO is deeply saddened by the news of the crash of Air Algerie flight AH 5017. On behalf of the international tourism community, UNWTO conveys its heartfelt sympathy to the family and friends of those onboard. |
23 July 2014 Addis Abeba Ethiopia |
Ethiopia - tourism key to promote development During an official visit to Ethiopia, UNWTO Secretary-General, Taleb Rifai, met with President Mulatu Teshome, Prime Minister Hailemariam Desalegn, Deputy Prime Minister Demeke Mekonnen, and Minister of Culture & Tourism, Amin Abdulkadir, to address the advancement of tourism in Ethiopia and the key role of the sector in the country's socio-economic development. (Addis Abeba, Ethiopia, 13-18 July 2014). |
22 July 2014 Madrid Spain |
UNWTO and Australia's Griffith University join forces to advance tourism knowledge Disseminating tourism knowledge, enhancing the expertise on tourism and climate change and advancing sustainable tourism in Asia and the Pacific are some of the areas of cooperation agreed between the Griffith Institute for Tourism (GIFT) and UNWTO (Madrid, Spain, 21 July 2014). |
21 July 2014 Madrid Spain |
International tourism: strong peak season anticipated Demand for international tourism remained strong in the first four months of 2014 according to the latest UNWTO World Tourism Barometer. International tourist arrivals worldwide grew by 5%, the same rate as during the full year 2013. Prospects for the current peak tourism season remain very positive with over 460 million tourists expected to travel abroad in the May-August 2014 period. |
18 July 2014 Madrid Spain |
UNWTO welcomes new Law on Antiquities and Heritage in Saudi Arabia UNWTO welcomes the new Saudi legislation aimed at protecting the country's antiquities and heritage as well as to give the Saudi Commission for Tourism and Antiquities (SCTA) the mandate to ensure its preservation. Heritage conservation is today fundamental to safeguard cultural legacy but also to promote and preserve key tourism assets which are central to differentiating destinations all around the world. |
18 July 2014 Madrid Spain |
UNWTO shocked by the tragic loss of Malaysia Airlines flight UNWTO is deeply shocked by the tragic crash of Malaysia Airline flight MH17. On behalf of the international tourism community, UNWTO conveys its heartfelt condolences to the families and friends of the victims. |
11 July 2014 Paris France |
UNWTO and France debate areas of further cooperation UNWTO Secretary-General, Taleb Rifai, and the French Minister of State in charge of Tourism, Fleur Pellerin, met to discuss the new tourism strategy of France as well as how to further strengthen the collaboration with UNWTO. Issues on the agenda included visa facilitation, taxation and the World Committee on Tourism Ethics (Paris, 30 June 2014). |
2 July 2014 Madrid Spain |
Spain to host UNWTO Conference on Religious Heritage and Tourism The city of Elche in Spain will host the UNWTO International Conference on Religious Heritage and Tourism. The Conference, to be held on 27-28 November 2014 in collaboration with the State Secretariat of Tourism of Spain, will highlight best practices in sustainable development of religious tourism and how this segment can contribute to product diversification. |
1 July 2014 Cartagena de Indias Colombia |
Tourism Ministers of the Americas commit to sustainable tourism Advancing public-private partnerships and incorporating sustainability in all tourism operations are cross-cutting challenges shared by the tourism authorities of the Americas. Gathered at the 57th Meeting of the UNWTO Commission for the Americas in Cartagena de Indias, delegations from 19 countries and around 100 private sector representatives took stock of the region's tourism and outlined the path to ensure that tourism's benefits contribute to the sustainable development of the region. (Colombia, 25 June 2014). |
23 June 2014 Madrid Spain |
World Tourism Day 2014: Celebrating tourism and community development 'Tourism and Community Development' is the theme of this year's World Tourism Day (WTD). The theme underscores the potential of tourism to promote opportunities for communities around the world, as well as the role that community engagement has in advancing sustainable tourism development. The official celebrations will be hosted by Mexico in the city of Guadalajara (27 September 2014). |


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In this paper, we describe how a mobile robot only controlled by visual information can retrieve particular goal locations in an open environment according to the level of associated motivations and to its current location. The interest of our control architecture relies in its simplicity and its robustness. Our robot does not need a precise map nor to learn all the possible positions in the environment to be able to navigate correctly. The neural control architecture used is inspired from neurobiological studies about the brain visual processes and the integration of those information for navigation behaviors. We emphasize the interest to go back and forth between robotics and biology. At last we show neurobiological modelization of the hippocampus has helped us to imagine a solution for scaling our architecture to more complex problems.


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SDN Members don't see this ad. (About Ads) hi everyone, i'm at that point where i'm deciding what i want to do with my life. my first choice as of now is urology, with orthopaedics lingering in the background. i've spent lots of time shadowing and getting surgical experience, but i'm still a little confused. one of the key things that i'm lacking experience in is the clinic side of things. does anyone have any opinion or information on what clinic life is like for urology? i have till midnight sunday before i have to confirm my 4th year electives...thanks in advance!
you get alot of clinic time with urology, probably similar to ENT. There are in-office prodecures you can do too during clinic: vasectomy, cystoscopy, prostate biopsy, transrectal ultrasound, etc. In fact, some older urologist will transition to an office-based practice
Hi, not sure if I can help much, but I'll try. Urology and ortho are VERY different fields. I'm assuming you must have done some 3rd year surgical rotations in both fields to be considering them.... which one did you enjoy more? I haven't had any ortho rotations (none are required at my school and I chose different surgical selectives) so I can't really comment on what life is like as an orthopedic surgeon or what their clinics are like. Urology is a varied specialty... I'm going to say probably moreso than ortho. But, like all specialities, you're going to end up doing a lot of the same thing over and over. Most of the urologists I've worked with have about 2 OR days a week. The rest of their time is spent in clinic or doing local procedures like vasectomies and cystos. Clinic can be anything from a woman with chronic pelvic pain and recurrent UTIs, to incontinence, stones, prostate ca, adrenal masses, renal cancer, hematuria, etc. Urology generally has home call and less emergency cases than most other surgical specialties. I'm not really sure what else to say... they are VERY different. Which one do you like better? Good luck! Try checking out this website to see if it helps http://www.urologymatch.com/
hi, thanks for the reply. i've spent some time doing both and enjoy both - hence the dilemma! with ortho, i really like the anatomy, the process of looking at radiographs to find the fractures, the clinical examination of patients with ortho issues, etc, however, i think that i may find the procedures a little repetitive after some time (and i've been told that lifestyle isn't too great with call etc). with urology, it's more the procedures that i enjoyed, as well as the opportunity for long term patient contact as well as more acute treatments. not to mention that in urology, the staff/residents i've worked with seem much more similar to me in personality as well as the lifestyle is great with minimal call etc. (except right now in my life i'd love to come in any time for interesting procedures etc, but i know that practically, family etc is important to me as well) any further thoughts? ps - thanks for the website, i've checked it out before and it's a great resource!
Hi, I don't really have much more to add... it sounds like you've experienced both and now you have to try to decide between the two. It's not easy to decide on a specialty, so I understand where you're coming from. I'm not completely familiar with the US residency match (I'm from Canada), but is there any way that you can do some ortho and urology electives and still hope to match to whichever one you like best? Ortho and urology are both quite competitive here and it's probably almost impossible to do that and still have a strong application. Are you allowed to double book yourself for electives? You could schedule overlapping ortho and urology for now and then figure out which one you want to go for and cancel the other electives. It's not the greatest thing to do, but if you really don't know... You mentioned that you think you would find ortho to be quite repetitive after a while. I think you will run into that with any specialty. It also depends on where you end up practicing. If you're in an academic centre, you might be the knee guy (or girl) or ankle guy or whatever, but in the community you might have a more varied practice (but maybe without the heavy hitting cases). Good luck


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Frank Leitz
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The following research project was funded in whole or in part by the Science and Technology Program. Frank Leitz is listed as the principal investigator, or primary researcher. Click the project's title to view more information.
Developing a deterministic model for predicting cleaning frequency due to inorganic scaling on reverse osmosis membranes
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2014 Summer Reading Program –
SIGN-UP begins Monday, June 16
Summer Reading 2014 – July 7 – August 11.
Independent Summer Reading! Get your books at the library.
READ! and Earn raffle tickets
ATTENTION 4th, 5th & 6th Grade students!!
Join our Summer Book Club – read this accelerated reader book –
Title: When Zachary Beaver Came to Town by Kimberly Willis Holt
(AR level: BL 4.5 Pts 6.0)
Pick up the book at the library, read it and then join us on
August 5 @ 2 pm. We will discuss the book and then we'll watch the movie version of the book. Snacks provided. You must read the book when you sign up (remember the movie is often different from the book). This will count as 1 book for your AR Summer Reading!
Books will be available at the library.
Chemistry Week
Monday, July 7 – Family Nite: Science Centers (chemistry stew) w/ craft: bring a t-shirt to decorate
Tuesday, July 8 – Drop-in craft: Scratch art
Thursday, July 10 at 2 pm – Ed Popielarczyk, magician, balloon artist
Senses Week
Monday, July 14 – Family Nite: Sensory Centers
Tuesday, July 15 – Drop-in craft: Make a sensory box
Wednesday, July 16 – Icky Sticky (sponsored by a Wachusett CFCE Grant)
Thursday, July 17 – Teaching Creatures: Animal Defenses, live animals presentation
Weather & Motion Week
Monday, July 21 – Family Nite: Lego Races
Tuesday, July 22 – Drop in craft: make a windchime
Thursday, July 24 – Truck Day: Trucks Around Town
Geology Week
Monday, July 28 – Family Nite: Rock Identification w/ craft: make a lava lamp
Tuesday, July 29 – Drop-in Craft: create stone art w/ rocks and model magic
Thursday, July 31 – TBA
Animal Week
Monday, August 4 – Second Chance Animal Shelter will visit (bring an item to donate!)
Tuesday, August 5 – Drop-in craft: make an animal puppet
Thursday, August 7 – Zoo in Forest Park, live animals presentation
Monday, August 11: End of Summer Reading: Pizza Party & Raffle Drawing 6 pm


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Bill Kenwright Presents Agatha Christie's Go Back For Murder
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Bill Kenwright Presents Agatha Christie's Go Back For Murder: Agatha Christie Theatre Company
Carla Le Marchant has learnt a disturbing family secret: her mother, Caroline Crale, died in prison after being convicted for killing her father.
Leaving an intriguing legacy in the form of a letter, Caroline professes her innocence and, believing this to be true; Carla becomes determined to clear her mother's name.
Enlisting the help of Justin Fogg; the son of her mother's defence lawyer, Carla searches out all the players from her tragic history and brings them back to the scene of the crime to uncover the truth.
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How should I handle my relatives who disapprove of breastfeeding?
My mom, sister, mother-in-law, and sister-in-law are all very unhappy with my decision to breastfeed my baby who is now 7 months old. Every time the subject comes up, they either make rude comments or give me dirty looks. My husband and I made the decision to breastfeed together, but I seem to take all the heat for it. How do I handle the criticism without fighting with anyone? I don't want to hurt my family's feelings but I feel this is the best choice for my baby.
Posted: 03/14/2000 by a BabyCenter Member
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Its not their baby.posted 10/07/2014 by HighRisk222
This is crazy to me! First of all, its the best thing that you can do for your baby. Secondly, its FREE. They are not the mother. It is your baby. I would tell them politely that you've made your decision and if they don't support it, their comments are not necessary.posted 08/23/2014 by ashleesm
You should ignore them. It has been scientifically proven that breastfeeding as long as you can is beneficial for the baby. There is nothing better than giving your child his/her natural sustainance. If I could have breastfed my little one as long as I could, I would have, but I'm one of those unfortunate women who has to work 3 hours away from her child 5 days a week and the stress and tiredness has affected my breast milk supply severely that my baby has had to rely mainly on formula :/posted 08/21/2014 by jellybelly01
ignore what they think, its good for your child. and if raising your child in a healthy and nurturing way offends them then they have problems. most parents I know breast feed till the child is two either in a bottle or from the boob it doesn't matter, your child will grow to be strong and smart and happy.posted 08/07/2014 by momofcece
JobandPay.composted 08/05/2014 by a BabyCenter Member
wow that is sad they are acting that way. You are the parent not them. I would ignore it if at all possible. Why the heck do they care anyways?? You are right breast is the best thing for baby so you go girl.posted 03/11/2010 by nikky212121
Keep it up!! I hate how this country has put formula before Breast. Formula should only be handed out when a mother CANT. Formula is bad its not what babies were ment to eat. You can do it! Your baby will only be a baby once and they grow so fast. My son will be 8 months tomorrow and we are still on pemo boob milk. I plan to breastfeed him until hes one and then he can get his milk from the cow. (My dads cow to be exact! Organic) :)posted 06/10/2014 by VKuntze
that is ridiculous and they need to get educatedposted 05/27/2014 by joel&jessijoy#1
They are ignorant . Point blank periodposted 05/14/2014 by Karyukaii
Tell them to F off. Breastfeeding is what every mother SHOULD be doing. Its better for the baby than any formula out there and they are in no position to make you feel otherwise. Sorry to be so blunt but they are obviously ignorant to be judging you for doing the best possible thing for your baby. Props to you for sticking to it. Its a shame how ill-informed people are about breastfeeding. You are doing the right thing. Dont let anyone convince you otherwise.posted 04/30/2014 by mlw808
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