text:
|Numéro de publication||US7927797 B2|
|Type de publication||Octroi|
|Numéro de demande||US 11/045,678|
|Date de publication||19 avr. 2011|
|Date de dépôt||28 janv. 2005|
|Date de priorité||28 janv. 2004|
|État de paiement des frais||Caduc|
|Autre référence de publication||CA2553833A1, CA2553833C, EP1735458A2, EP1735458B1, US20050227264, US20110177587, WO2005073410A2, WO2005073410A3|
|Numéro de publication||045678, 11045678, US 7927797 B2, US 7927797B2, US-B2-7927797, US7927797 B2, US7927797B2|
|Inventeurs||John R. Nobile, William L. Lee, John H. Leamon|
|Cessionnaire d'origine||454 Life Sciences Corporation|
|Exporter la citation||BiBTeX, EndNote, RefMan|
|Citations de brevets (69), Citations hors brevets (31), Référencé par (46), Classifications (40), Événements juridiques (5)|
|Liens externes: USPTO, Cession USPTO, Espacenet|
This application claims the benefit under 35 U.S.C. §119(e) of U.S. provisional patent application No. 60/540,016, filed Jan. 28, 2004. This application is also related to the following application Nos. 60/443,471, filed Jan. 29, 2003; 60/465,071, filed Apr. 23, 2003; 60/476,313, filed Jun. 6, 2003; 60/476,504, filed Jun. 6, 2003; U.S. Ser. Nos. 60/476,592, filed on Jun. 6, 2003; 60/476,602, filed Jun. 6, 2003; and 60/497,985, filed Aug. 25, 2003. All patent and patent applications in this paragraph are hereby fully incorporated by reference.
This application also incorporates by reference the following copending U.S. patent application Ser. Nos. 10/767,899, filed Jan. 28, 2004; 10/767,894, filed Jan. 28, 2004; and 10/767,779, filed Jan. 28, 2004.
Embodiments of the present invention relate to methods and systems for clonally amplifying nucleic acid templates from a single copy number to quantities amenable for sequencing, as well as methods and systems for continuous flow PCR using emulsion and solid support for immobilizing amplified nucleic acids.
The ability to amplify a plurality of nucleic acid sequences, such as a genomic library or a cDNA library, is critical given the current methods of sequencing. Current sequencing technologies require millions of copies of nucleic acid per sequencing reaction, therefore, amplification of the initial DNA is necessary before genomic sequencing. Furthermore, the sequencing of a human genome would require tens of millions of different sequencing reactions.
Current techniques and systems for in vitro genome amplification involve laborious cloning and culturing protocols that have limited the utility of genomic sequencing. Other techniques, such as PCR, while fast and reliable, are unable to amplify a mixture of different fragments of DNA a genome in a representative and clonal fashion.
While random primed PCR can be easily engineered to amplify a plurality of nucleic acids in one reaction, this method is not preferred because the amplified product will be a mixture of different DNA fragments from the library. In addition, in a random PCR environment starting with a plurality of fragments, some DNA sequences are preferentially amplified at the expense of other sequences such that the amplified product does not represent the starting material. This problem with PCR may be overcome if each individual member of a library is amplified in a separate reaction.
However, this approach may be impractical if many thousands of separate reaction tubes are required for the amplification process, as a genomic library or cDNA library may include more than 1,000,000 fragments. Individual amplification of each fragment of these libraries in separate, conventional reaction tube is not practical.
The present invention provides for methods and systems for amplifying a plurality of nucleic acids (e.g., each sequence of a DNA library, transcriptome, or genome) in a rapid and economical manner using, for example, a means for encapsulating a plurality of DNA samples effectively individually in a microcapsule of an emulsion (i.e., a “microreactor”), performing amplification of the plurality of encapsulated nucleic acid samples simultaneously, and releasing the amplified plurality of DNA from the microcapsules for subsequent reactions.
Preferably, in some embodiments of the invention, a plurality of such microreactors include at least one capture bead (and preferably a single bead). Each capture bead is preferably designed to have a plurality of oligonucleotides that recognize (i.e., are complementary to) a portion of a nucleic acid template, and the amplification copies of this template. It is preferred that any one capture bead contain only one unique nucleic acid species.
Embodiments of the present invention provide methods and systems for performing continuous flow amplification, specifically, encapsulated continuous flow amplification. Such embodiments may be used with thermal or isothermal amplification reactions, for example, PCR, rolling circle amplification, whole genome amplification, nucleic acid sequence-based amplification, and single strand displacement amplification. In preferred embodiments, the apparatus employs steady state heat generation and transfer elements and cross-flow emulsion droplet generation as described in detail herein.
Accordingly, in one embodiment of the invention, a method for amplifying genetic material includes providing a water-in-oil emulsion in a continuous flow wherein the emulsion comprises a plurality of water-based droplets comprising microreactors. The plurality of the microreactors may each include one or more species of nucleic acid templates, and sufficient reagents to amplify the copy number of one of the nucleic acid templates. The method may also include thermally processing the emulsion by flowing it across stationary controlled temperature zones to amplify nucleic acid templates by polymerase chain reaction.
In another embodiment of the present invention, an apparatus for amplifying genetic material includes at least one fluid delivery device, at least one first temperature zone to cycle a plurality of microreactors each including one or more species nucleic acid templates to a first temperature, at least one second temperature zone to cycle the plurality of microreactors to second temperature lower than the first temperature, a first conduit for flowing at least a stream of oil therein from a first reservoir and a second conduit for flowing at least a water based PCR solution from a second reservoir out of an orifice and into the first conduit creating a water-in-oil emulsion. The PCR solution upon entering the first conduit comprises a plurality of droplets comprising the microreactors for performing polymerase chain reactions. A plurality of the microreactors each include one or more species of nucleic acid template.
In another embodiment of the invention, a cross-flow emulsification apparatus includes a first inlet for receiving an oil flow from a first conduit, an outlet for directing a water-in-oil emulsion out of the apparatus, a second inlet for receiving a water based PCR amplification reaction mixture from a second conduit and an orifice for delivering PCR reaction mixture from the second conduit into the first conduit, to form a plurality of water-in-oil droplets comprising microreactors. A plurality of the microreactors each include one or more nucleic acid templates and sufficient PCR amplification reaction mixture to produce a plurality of copies of nucleic acid template.
In another embodiment of the present invention, an apparatus for amplifying genetic material includes a water-in-oil emulsion in a continuous flow wherein the emulsion comprises a plurality of water droplets comprising microreactors. A plurality of the microreactors may include a single bead capable of capturing one or more nucleic acid templates, and sufficient reagents to amplify the copy number of the one or more nucleic acid templates. The apparatus may also include thermal processing means for thermally processing the emulsion to amplify nucleic acid templates by polymerase chain reaction.
In another embodiment of the present invention an emulsion generator including an emulsion oil supply, at least one syringe including a body and a plunger for dispensing a mixture for emulsifying into the emulsion oil, a cross-flow emulsification device for emulsifying the mixture, the device including an input attached to the output of the syringe, a syringe pump including an actuator capable of oscillating the plunger of the at least one syringe micrometer distances at a predetermined frequency along a length of travel of the plunger within the syringe body of the at least one syringe.
In another embodiment of the present invention, a method for substantially reducing clogging of a nozzle in syringe pump includes providing a syringe pump having at least one syringe including a body, a plunger having a plunger axis and an exit nozzle, the body for dispensing a mixture of micron or less sized particles suspended in a medium, and oscillating the plunger of the syringe along the axis of the plunger for micrometer distances at a predetermined frequency along a length of travel of the plunger within the syringe body.
In another embodiment of the present invention, an emulsion generator includes an emulsion oil supply, at least one syringe including a body and a plunger for dispensing a mixture for emulsifying into the emulsion oil, a magnetically-attractive mixing element disposed in the body of the syringe, a cross-flow emulsification device for emulsifying the mixture, the device including an input attached to the output of the syringe and a device capable of moving an external magnetic force axially along body of the syringe while in close proximity to the syringe body.
In another embodiment of the present invention an emulsion generator includes an emulsion oil supply, at least one syringe including a body and a plunger for dispensing a mixture for emulsifying into the emulsion oil, a magnetically-attractive mixing element disposed in the body of the syringe, a cross-flow emulsification device for emulsifying the mixture, the device including an input attached to the output of the syringe and a rotating drum having a magnet helically wound along the surface of the drum. The surface of the drum is positioned adjacent the body of the syringe.
In another embodiment of the present invention, a syringe pump includes an area for receiving at least one syringe, where the syringe includes a body and a plunger having a plunger axis. The syringe may be used for dispensing a mixture for emulsification into an emulsion oil. The syringe pump may also include an actuator capable of oscillating the plunger of the at least one syringe along the plunger axis micrometer distances at a predetermined frequency along a length of travel of the plunger within body of the at least one syringe.
In another embodiment of the present invention, a syringe pump includes an area for receiving at least one syringe, where the syringe includes a body and a plunger having a plunger axis. The syringe may be used for dispensing a mixture for emulsification into emulsion oil. The syringe pump may also include a magnetically attractive mixing element disposed in the body of the syringe and a rotating drum having a magnet helically wound along the surface of the drum, wherein the surface of the drum is positioned adjacent the body of the syringe.
Other objects, advantages, features and benefits of the invention will become more readily apparent by reference to the attached drawings and following detailed description of the invention.
Brief Overview of Bead Emulsion Amplification
Bead emulsion amplification may be performed by attaching a template (e.g., DNA template) to be amplified, to a solid support, preferably in the form of a generally spherical bead. The bead is linked to a large number of a single primer species that is complementary to a region of the template DNA and the amplification copies of this template. Alternately, the bead is linked to chemical groups (e.g., biotin) that can bind to chemical groups (e.g., streptavidin) included on the template DNA and amplification copies of this template. See WO2004069849, herein incorporated by reference.
The beads are suspended in aqueous reaction mixture and then encapsulated in a water-in-oil emulsion. The template DNA may be bound to the bead prior to emulsification, or the template DNA is included in solution in the amplification reaction mixture.
The emulsion may be composed of discrete aqueous phase microdroplets (i.e., microreactors, see above), e.g., averaging approximately 60 to 200 μm in diameter, enclosed by a thermostable oil phase. Each microreactor contains, preferably, sufficient amplification reaction solution (i.e., the reagents necessary for nucleic acid amplification). An example of an amplification reaction solution would be a PCR reaction mixture (polymerase, salts, dNTPs; and may also preferably include a pair of PCR primers (primer A and primer B). In some cases, the template DNA is included in the reaction mixture. A subset of the microreactor population preferably includes microreactors having a single DNA bead preferably with an attached nucleic acid template. This subset of microreactors is the basis for the amplification in some of the preferred embodiments of the present application. In one embodiment, the remaining microreactors which do not contain template DNA will not participate in amplification.
PCR amplification and PCR primers may be present in an asymmetric ratio such as 8:1 or 16:1 (i.e., 8 or 16 of one primer to 1 of the second primer) to perform asymmetric PCR. The primer species that may be used in the lower concentration level is the same primer species that may be immobilized on the bead. This will increase the probability that an amplified copy of the template DNA will anneal to the bead. The ratio of PCR primers may also be substantially equal for normal PCR. The amplification reaction, such as PCR, may be performed using any suitable method.
After PCR, the beads containing the immobilized amplified DNA may be recovered. The emulsion may be broken to recover the beads. The immobilized product may be rendered single stranded by denaturing (by heat, pH etc.) which removes the complimentary A strand. The A primers are annealed to the A′ region of immobilized strand, and the beads containing the immobilized strands are loaded with sequencing enzymes, and any necessary accessory proteins. The beads are then sequenced using recognized pyrophosphate techniques (described, e.g., in U.S. Pat. Nos. 6,274,320, 6258,568 and 6,210,891, incorporated in toto herein by reference).
In a preferred embodiment, the nucleic acid template to be amplified by bead emulsion amplification is a population of DNA such as, for example, a genomic DNA library or a cDNA library. It is preferred that each member of the DNA population have a common nucleic acid sequence at the first end and a common nucleic acid sequence at a second end. This can be accomplished, for example, by ligating a first adaptor DNA sequence to one end and a second adaptor DNA sequence to a second end of each member of the DNA population. The nucleic acid template may be of any size amenable to in vitro amplification (including the preferred amplification techniques of PCR and asymmetric PCR). In a preferred embodiment, the template is about 150 to 750 bp in size, such as, for example about 250 bp in size.
Binding Nucleic Acid Template to Capture Beads
A single stranded nucleic acid template to be amplified may be attached to a capture bead. The amplification copies of the nucleic acid template may also be attached to a capture bead. As non-limiting examples, these attachments may be mediated by chemical groups or oligonucleotides that are bound to the surface of the bead. The nucleic acid (e.g., the nucleic acid template, amplification copies, or oligonucleotides) may be attached to the solid support (e.g., a capture bead) in any manner known in the art.
According to the present invention, covalent chemical attachment of a nucleic acid to the bead can be accomplished by using standard coupling agents. For example, water-soluble carbodiimide can be used to link the 5′-phosphate of a DNA sequence to amine-coated capture beads through a phosphonamidate bond. Alternatively, specific oligonucleotides can be coupled to the bead using similar chemistry, and then DNA ligase can be used to ligate the DNA template to the oligonucleotide on the bead. Other linkage chemistries to join the oligonucleotide to the beads include the use of N-hydroxysuccinamide (NHS) and its derivatives, for example.
In an exemplary method, one end of a linker may contain a reactive group (such as an amide group) which forms a covalent bond with the solid support, while the other end of the linker contains a second reactive group that can bond with the oligonucleotide to be immobilized. In a preferred embodiment, the oligonucleotide is bound to the DNA capture bead by covalent linkage. However, non-covalent linkages, such as chelation or antigen-antibody complexes, may also be used to join the oligonucleotide to the bead.
As non-limiting examples, oligonucleotides can be employed which specifically hybridize to unique sequences at the end of the DNA fragment, such as the overlapping end from a restriction enzyme site or the “sticky ends” of cloning vectors, but blunt-end linkers can also be used. These methods are described in detail in U.S. Pat. No. 5,674,743. It is preferred that the beads will continue to bind the immobilized oligonucleotide throughout the steps in the methods of the invention.
Each capture bead is preferably designed to have a plurality of oligonucleotides that recognize (i.e., are complementary to) a portion of the nucleic template, and the amplification copies of this template. It is preferred that any one capture bead contain only one unique nucleic acid species.
The beads used herein may be of any convenient size and fabricated from any number of known materials. Example of such materials include: inorganics, natural polymers, and synthetic polymers. Specific examples of these materials include: cellulose, cellulose derivatives, acrylic resins, glass, silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene or the like (as described, e.g., in Merrifield, Biochemistry 1964, 3, 1385-1390), polyacrylamides, latex gels, polystyrene, dextran, rubber, silicon, plastics, nitrocellulose, natural sponges, silica gels, control pore glass, metals, cross-linked dextrans (e.g., Sephadex™) agarose gel (Sepharose™), and other solid phase supports known to those of skill in the art. In preferred embodiments, the capture beads are beads approximately 2 to 100 μm in diameter, or 10 to 80 μm in diameter, most preferably 20 to 40 μm in diameter. In a preferred embodiment, the capture beads are Sepharose beads.
Capture beads with or without attached nucleic acid template may be suspended in a heat stable water-in-oil emulsion. Furthermore, the size of the microreactors may be adjusted by varying the flow rate and speed of the components. Additionally droplet size can also be varied by changing the viscosity of the emulsion oil, and also by the using different orifice sizes in the cross-flow emulsion generating part.
Various emulsions that are suitable for biologic reactions are referred to in Griffiths and Tawfik, EMBO, 22, pp. 24-35 (2003); Ghadessy et al., Proc. Natl. Acad. Sci. USA 98, pp. 4552-4557 (2001); U.S. Pat. No. 6,489,103 and WO 02/22869, each fully incorporated herein by reference. It is noted that Griffiths et al., (U.S. Pat. No. 6,489,103 and WO 99/02671) refers to a method for in vitro sorting of one or more genetic elements encoding a gene products having a desired activity. This method involves compartmentalizing a gene, expressing the gene, and sorting the compartmentalized gene based on the expressed product. In contrast to the present invention, the microencapsulated sorting method of Griffith is not suitable for parallel analysis of multiple microcapsules/microreactors because their nucleic acid product is not anchored and cannot be anchored. Since the nucleic acids of Griffiths are not anchored, they would be mixed together during demulsification.
The emulsion is preferably generated by adding beads to an amplification solution. As used herein, the term “amplification solution” means the sufficient mixture of reagents that is necessary to perform amplification of template DNA. One example of an amplification solution, a PCR amplification solution, is provided in the examples below. It will be appreciated that various modifications may be made to the amplification solution based on the type of amplification being performed and whether the template DNA is attached to the beads or provided in solution. The oil used may be supplemented with one or more biocompatible emulsion stabilizers including Agrimer AL22 and other recognized and commercially available suitable stabilizers.
In preferred aspects, the emulsion is heat stable to allow thermal processing/cycling, e.g., to at least 94° C., at least 95° C., or at least 96° C. Preferably, the droplets formed range in size from about 5 microns to 500 microns, more preferably, from about 50 to 300 microns, and most preferably, from about 100 to 150 microns. Advantageously, cross-flow emulsion generation allows for control of the droplet formation, and uniformity of droplet size.
The microreactors should be sufficiently large to encompass sufficient amplification reagents for the degree of amplification required. However, the microreactors should be sufficiently small so that a sufficient number of microreactors, up to about 20,000,000 or more, each containing effectively a single member of a DNA library, can be supplied from a small number of conventionally available syringes that can fit together on a syringe pump. Notably, the use of microreactors allows amplification of complex mixtures of templates (e.g., genomic DNA samples or whole cell RNA) without intermixing of sequences, or domination by one or more templates (e.g., PCR selection bias; see, Wagner et al., 1994, Suzuki and Giovannoni, 1996; Chandler et al., 1997, Polz and Cavanaugh, 1998).
With the limitations described above, the optimal size of a microreactor may be on average 100 to 200 microns in diameter. Microreactors of this size would allow amplification of a DNA library comprising about 18,000,000 members supplied to the emulsion generator in a volume of 9 mls contained in three 3 ml syringes.
After encapsulation of the bead and PCR solution and template DNA in the micro-reactor, the template nucleic acid may be amplified, while attached (preferably) or unattached to beads, by any suitable method of amplification including transcription-based amplification systems (Kwoh D. et al., Proc. Natl. Acad Sci. (U.S.A.) 86:1173 (1989); Gingeras T. R. et al., WO 88/10315; Davey, C. et al., EP Publication No. 329,822; Miller, H. I. et al., WO 89/06700), “RACE” (Frohman, M. A., In: PCR Protocols: A Guide to Methods and Applications, Academic Press, NY (1990)) and one-sided PCR (Ohara, O. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86.5673-5677 (1989)). Still other methods such as di-oligonucleotide amplification, isothermal amplification (Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)), Nucleic Acid Sequence Based Amplification (NASBA; see, e.g., Deiman B et al., 2002, Mol Biotechnol. 20(2):163-79), whole-genome amplification (see, e.g., Hawkins T L et al., 2002, Curr Opin Biotechnol. 13(1):65-7), strand-displacement amplification (see, e.g., Andras S C, 2001, Mol Biotechnol. 19(1):29-44), rolling circle amplification (reviewed in U.S. Pat. No. 5,714,320), and other well known techniques may be used in the present invention.
In a preferred embodiment, DNA amplification is performed by PCR. PCR according to the present invention may be performed by encapsulating the target nucleic acid with a PCR solution comprising all the necessary reagents for PCR. Then, PCR may be accomplished by exposing the emulsion to any suitable thermal processing regimen known in the art. In a preferred embodiment, 30 to 60 cycles, and preferably about 60 cycles, of amplification are performed. It may be desirable, but not necessary, that following the amplification procedure, there may be one or more hybridization and extension cycles, which comprise a similar melting time but a longer extension time, following the cycles of amplification. Routinely, the template DNA is amplified until typically at least two million to fifty million copies, preferably about ten million to thirty million copies of the template DNA are immobilized on each bead.
In some embodiments, the method of the invention employs continuous flow PCR to amplify the nucleic acid template. Various methods of continuous flow PCR have been reported in, e.g., Park et al., 2003, Anal. Chem. 75:6029-6033; Curcio and Roeraade, 2003, Anal. Chem. 75:1-7; Chiou et al., 2001, Anal. Chem. 73:2018-2021; U.S. Pat. No. 6,207,031; U.S. App. Pub. 2001/0020588; Lagally et al., 2001, Anal. Chem. 73:565-570; U.S. Pat. Nos. 6,361,671, 6,284,525, 6,132,580, 6,261,431, 6,045,676, 6,143,152, 5,939,312; U.S. App. Pub. 2002/0068357; Schneegas et al., 2001, Lab on a Chip 1:42-49; Kopp et al., 1998, Science 280:1046-1049; Nakano et al., 1994, Biosci. Biotech. Biochem. 58:349-352; and Larzul in U.S. Pat. No. 5,176,203, all of which are incorporated herein by reference. Advantageously, continuous flow PCR greatly reduces sample handling and reaction times, while it increases amplification specificity. However, previous flow systems utilized serial slugs, i.e., slugs of reagent that completely fill the diameter of the flow tube, that are separated by similar full slugs of air and oil. In contrast, embodiments of the present invention are directed to the a water-in-oil emulsion used in conjunction with a continuous flow PCR. The water-in-oil emulsion comprises microreactors, allowing clonal amplification of a large population of nucleic acids. The microreactors are about 10 to 50 times smaller than the diameter of the flow tube so that a very large number of them are present in the flow stream. For example, a 2 mm diameter flow tube can carry 2,000 microreactors per cm of length.
The continuous flow PCR methods of the invention can be used to amplify the sequences of an entire genome or transcriptome on a single instrument in less than half the time required for traditional thermal processing. Continuous flow of the emulsion across a solid state heat transfer element permits efficient and rapid (e.g., 60 second) reaction cycle. A 60 cycle amplification for example, would take 1 hour. In various embodiments, the nucleic acid template can be diluted to obtain effectively one copy of delivered template per microreactor, and a final yield of 1,000,000 to 10,000,000 template copies per bead. As examples, the continuous flow methods of the invention can be used with thermal amplification reactions (e.g., PCR) or isothermal reactions (e.g., rolling circle amplification, whole genome amplification, NASBA, strand displacement amplification, and the like).
In one aspect, the method of the invention is performed using a system for continuous flow amplification, e.g., continuous flow PCR amplification. This system includes a means for forming an emulsion of an amplification reaction mixture in a stabilized biocompatible oil.
The plurality of microreactors may then be thermally processed via a thermal processor 112 to amplify the DNA template. After amplification, the beads (each containing the amplified nucleic acid), may be filtered out of the emulsion flow via a bead filter 314, and thereafter processed for subsequent sequencing.
The emulsion oil is supplied to the cross-flow emulsifier 210 (see also 106,
As shown in
The emulsifier 400 also includes a second inlet 410 of a second conduit 412 for directing amplification reaction/bead mixture into the apparatus. A tubular orifice 414 is provided at a terminus of the second conduit, and is open to the narrowed area 406 of the first conduit. The orifice preferably includes a diameter of between about 10 um to about 200 μm, and more preferably between 75 μm and 150 μm, and most preferably about 120 μm. Preferably, the second inlet, conduit and orifice are provided in a vertical arrangement relative to the preferred horizontal arrangement of the first conduit (i.e., the first conduit and second conduit/orifice may be orthagonal to one another), although any orientation can work. The orifice enables a plurality of amplification reaction mixture droplets (i.e., microreactors), to be created as the reaction mixture enters the oil flow. A plurality of such microreactors each preferably include on average a single bead and an effective single nucleic acid template.
With reference to
Accordingly, when the emulsion oil enters a respective injection tee, it enters a progressively narrower region, and is thus accelerated to a higher velocity (e.g., 30 times its initial velocity). The PCR/bead solution (i.e., an aqueous phase material) is then injected at preferably a constant and controlled rate, preferably between about 5 μl-100 μl per minute, and preferably about 20 μl/minute. One of skill in the art will appreciate that other relative flowrates for the two component fluid sources may be used in accordance, for example, with the specifications of a thermal processing device for PCR amplification.
The shearing force of the high velocity oil breaks off the PCR/bead stream into individual droplets as it is being injected, each preferably including a single bead. As the newly formed droplets move downstream, the velocity of the flow may be gradually reduced in a diffuser area (see partial diffuser area 522,
The examples of injection tees illustrated in
The PCR/bead mixture is provided to the injection tee using a syringe pump, an example of which is illustrated in
To insure the beads are evenly distributed throughout the solution within each syringe a mixing mechanism may also be included with the syringe pump. As shown in
The magnets 626 in the rotating element are preferably oriented so that the fields are directed out radially from the rotating element. Preferably, a majority (and most preferably, all) of the magnets have the same polarity orientation.
As the helically mounted line of magnets 626 pass by the body of each syringe, a magnetic ball 632 a, 632 b, 632 c, included inside each syringe body is moved from its lowest position adjacent the plunger 634 toward a higher position, which may be adjacent the nozzle area 636, successively, higher and higher by each magnet. The ball is released once the highest magnet in the helical series moves away from the syringe body and then drops to the bottom (i.e., adjacent the plunger) of the syringe. This motion of the mixing ball will occur regardless of where the syringe plunger is located. The frequency and velocity of the mixing ball may be controlled by the rotational speed of the rotating element. Preferably, as the syringe becomes less full, the ball moves more quickly to accomplish the same mixing effect. More than one helical pattern of magnets may be used to allow different mixing rates and displacements as the plunger moves through different areas of the syringe. For example, it is desirable that the mixing action does not disturb the injection tee, so one series of magnets may start at the lowest point of the plunger and only go part way up the syringe body, while a second line of magnets may start at the middle and continue to the top. The lower magnets would work when the plunger is low, and the upper portion of the magnets would only work when the plunger is in the upper range of its motion. Another variation is for the helix of magnets to go both upward and downward, thereby controlling the motion throughout and allowing the mechanism to work in any orientation.
To insure the beads do not clog the nozzle in the mixing tee, some embodiments of the present invention make use of a anti-clogging device. More particularly, some embodiments of the present include a sonic (vibratory), anti-clogging mechanism (
Without the use of the sonic actuators according to the present invention, large amounts of viscosity enhancers and surfactants would be required to prevent particles from clogging the nozzles, and such a system would still only be marginally reliable. Another important function of the vibratory mechanical excitation of the syringe plungers is that it prevents the rubber syringe plungers from sticking to the internal body of the syringe. If this happens, a slip-stick intermittent motion of the syringe plunger will occur, causing the bead/PCR solution to be fed at a variable rate, causing droplet size control to be impossible.
The flow exiting from the emulsifier, via a conduit (for example) may be then run through a thermal processing device, which exposes the continuous flow to alternate zones of a higher temperature and lower temperature (e.g., a heating zone and a cooling zone) for PCR amplification. Examples of such thermal processing devices are illustrated in
For example, a stainless steel conduit, including an inlet 702 and an outlet 704, containing the emulsified flow (e.g., 400 as shown in
Alternatively, the second temperature zone may also include a heating element, since the zone typically, for PCR amplification, maintains a temperature of between approximately 60-70 degrees C., which is considerable higher than room temperature of 23 degrees C. Such an example is shown in the thermal processor illustrated in
In this example, the thermal processing device 800 allows alternating sections of a conduit 802 to be positioned adjacent opposed (for example) linear first temperature 804 and second temperature 806 zones. One or more thermoelectric heat transfer elements 808 of the thermal processing device may provide heat to the continuous flow along portions of the conduit. Resistance heaters 810 may be positioned adjacent the second temperature zone 806, so that the conduit sections positioned adjacent the second temperature zone is maintained at, for example, between a temperature of, for example, 60-70 degrees C. The second temperature may maintained by both adding heat from the heating elements when required, and also by removing heat in the second zone, using (for example) a fan to move air across the second zone. Another feature that may be included is an exposed area of conduit between the first and second temperature zone, which allows the conduit to be directly cooled by the fan, rather than via the second temperature zone block.
In this example, the resistance heaters may also provide thermal energy for the first temperature zone. Blocks 812 may be used as thermal conductors to conduct heat to and from the various zones and to and from the conduit, and may be fabricated from any material useful for heating, e.g., metal such as aluminum, copper, and the like. The blocks may be designed to be a precise fit around the conduit so that thermal energy may be efficiently transferred between the blocks and the conduit. A thermal grease may be added between these two elements to further improve the thermal conductivity of the interface. Insulation 814 may also be used to help maintain temperature of the high temperature zone, or any other area of the thermal processing device. In one example, the conduit may extend 46 cm for each amplification cycle, for a total of 35 cycles. In another example, the conduit is 67 cm for each cycle, and a total of 60 cycles are used. The conduit is made in groups of five cycles, so that sufficient fasteners may be used to ensure that the blocks are tightly clamped around the conduit. To allow a pre-heating step, an additional length of conduit may be added in the beginning that is exposed only to the high temperature zone. This may be included for the purpose of activating an enzyme required for PCR amplification. Further still, a distal end of the conduit may be adapted to allow for sample collection, e.g., into a bead filtering device or a collection container.
For PCR, the temperature in the first temperature zone of between 90 and 100 degrees C. may be used to melt duplex nucleic acid (e.g., 94° C.), while the 60-70 degrees C. temperature of the second temperature zone is chosen for primer annealing and extension (e.g., 65° C.), for example.
While the delivery of the emulsion components can be accomplished by any manual or automatic delivery means, preferably a pump system is used. As illustrative examples, delivery can be obtained by various pumps, including syringe pumps and mechanical pumps, e.g., HPLC pumps (see, e.g., Gilson, Inc., Middleton, Wis.; ESA, Inc., Chelmsford, Mass.; Jasco Inc, Easton, Md.). The preferred means is a rotary annular gear pump.
Exemplary heating devices for the apparatus include, but are not limited to, cartridge heaters (see, e.g., Omega Engineering, Inc., Stanford, Conn.; Delta-t Max, Greenland N.H.), resistive heaters (see, e.g., Minco Products, Inc., Minneapolis, Minn.), and thermoelectric heaters, including Peltier devices (see, e.g., Ferrotec, Nashua N.H.). In various aspects, the heating devices for the apparatus can be embedded in the heating blocks or mounted on the surface of the blocks. Temperature monitors may also be used with the apparatus, including real-time proportional temperature controllers, PID (proportional, integral, and derivative) digital controllers, in combination with temperature sensing elements such as thermocouples, thermistors, or any other suitable device (see, e.g., Watlow Electric Mfg. Co., St. Louis, Mo.).
The conduit material may be fabricated out of any compatible tubing material for amplification (in particular, thermal amplification), such as stainless steel, Polytetrafluoroethylene (PTFE; e.g., Teflon), and fused silica. Preferably, stainless steel tubing is used for its thermal conductivity and corrosion resistance.
It will be understood that other means for controlling amplification for the apparatus are also possible. For example, fluids can be circulated from constant temperature reservoirs, in particular, hot oil baths (see, e.g., Nakano et al., 1994, Biosci. Biotech. Biochem. 58:349-352), and hot water baths (see, e.g., Curcio and Roaeraade, 2003,Anal. Chem. 75:1-7). In addition, it is possible to perform continuous flow amplification on the surface of a chip (see, e.g., Kopp et al., 1998, Science 280:1046-1049; Schneegas et al., 2001, Lab on a Chip 1:42-49). For example, a silicon or glass chip can be modified to include thin film transducers to heat different sections of the chip to different temperatures. Alternatively, a chip can be placed across a row of heating blocks, where each block is heated to a different temperature. The heated sections of the chips can allow for denaturation (e.g., 95° C.), primer annealing (e.g., 58° C. or 60° C.), and primer extension (e.g., 72° C. or 77° C.) steps in the amplification reaction. In addition, fluid channels may be added to the chip (e.g., by etching, molding, imprinting, or adhesives) to allow for buffer and sample input, temperature cycling, and product output. The buffers and samples can be delivered, for example, by precision syringe pumps, and the amplification products can be collected into microfuge tubes, microwells, or other reservoirs. These methods, however, do not provide for the separate amplification of large numbers (thousands or millions) of different DNA fragment templates, which is the critical advantage of this invention.
Following amplification of the nucleic acid template and the attachment of amplification copies to the bead, the beads must be recovered. If a filter element is at the exit of the first conduit, the filter may be removed from the system, and beads may be back-flushed out of the filter using reverse flow. The beads may alternatively be washed & processed while they are still in the filter, by attaching the filter to a syringe with the bead side exposed to the syringe chamber, and pulling and pushing various wash reagents through the filter and in and out of the syringe.
Alternatively, the emulsion exiting the flow system may be collected in a vessel, and subsequently spun in a centrifuge, which will leave the beads at the bottom provided they are denser than the oil. The oil may then be removed from above the beads, and the beads my be recovered from the bottom of the vessel. This procedure may also be used without the centrifuge, if sufficient time is allowed for the beads to settle by gravity.
Purifying the Beads
After PCR amplification, the beads may be isolated from the microreactors and used for sequencing. The sequencing steps are preferably performed on each individual bead. However, this method, while commercially viable and technically feasible, may not be most effective because a portion of the beads will be “negative” beads (i.e., beads without amplified nucleic acid attached). This is because the DNA template material is delivered to the PCR solution or the beads by dilution, and it is inevitable that at least some of the beads do not get a starting copy for amplification. In such cases, an optional process outlined below may be used to remove negative beads prior to distribution onto multiwell (e.g., picoliter) plates.
This example describes preparation of a population of beads that preferably have only one unique nucleic acid template attached thereto. Successful clonal amplification depends on the delivery of a controlled number of template species to each bead. Delivery of excess species can result in PCR amplification of a mixed template population, preventing generation of meaningful sequence data while a deficiency of species will result in fewer wells containing template for sequencing. This can reduce the extent of genome coverage provided by the sequencing phase. As a result, it is preferred that the template concentration be accurately determined through replicated quantitation.
Template Quality Control
The success of the Emulsion PCR reaction is related to the quality of the template species. Regardless of the care and detail paid to the amplification phase, poor quality templates will impede successful amplification and the generation of meaningful sequence data. To prevent unnecessary loss of time and money, it is important to check the quality of the template material before initiating the PCR phase of the process. Preferably, the template library should pass two quality control steps before it is used in Emulsion PCR. Its concentration and the distribution of products it contains should be determined. Ideally, the library should appear as a heterogeneous population of fragments with little or no visible adapter dimers (e.g., ˜90 bases). Also, amplification with PCR primers should result in a product smear ranging, for example, from 300 to 500 bp. Absence of amplification product may reflect failure to properly ligate the adaptors to the template, while the presence of a single band of any size may reflect contamination of the template.
Continuous Flow PCR Amplification
A linear emulsifier included an internal diameter of 300 μm for an oil passage and an internal diameter of 120 μm for the bead/PCR solution outlet (see
Solution Phase Reaction Mix:
1× Hi Fl Buffer
1 mM dNTPs
2.5 mM MgSO4
1 uM forward Primer (MMP 1a)
1 uM Reverse Primer (MMP 1b)
0.15 U/ul Hi Fi Taq
Added 3 and 30 Copies of TF7 per nl PCR Mix
For Bead experiment used same reaction mix only added 0.333% 4M weight PEO and added 3,600 copeis/nl of TF7 into the solution. Added beads at 1 bead/nl.
Copy per nl
Pyrosequencing of Beads from Reaction Showed Clear TF7 Sequence.
The total amplification obtained was 50,000,000×, corresponding to 1.66× amplification per PCR cycle.
Another Example of Emulsion Flow PCR
The PCR amplification mixture used contained 1× High Fidelity Buffer (60 mM Tris-SO4 pH 8.9, 18 mM Ammonium Sulfate, (Invitrogen)), 1 mM dNTPs (Pierce), 0.625 mM forward primer, 0.078 mM reverse primer (IDT), 0.25% agrimer AL10-LC (ISP Technologies), 5% PEG-8000 (Acros), 0.02% BSA (Sigma), 0.003 U/ul inorganic pyrophosphotase (NEB), 0.15 U/ul Platinum High Fidelity Taq (Invitrogen).
The library of interest, E. coli, was added in three replicates to 1.8 million capture beads in a minimal volume and resuspended by vortexing. This mixture was then added to 900 μl of the PCR mixture. This solution was then loaded into a 1 ml syringe that contained an 4.1 mm plastic coated magnetic mixing ball. Three identical syringes were then loaded in series onto the “Flow PCR Unit”.
The emulsion flow PCR system included an internal diameter of 300 μm for an oil passage and an internal diameter of 120 μm for the bead/PCR solution outlet (see
2.42 million beads were recovered from the filter and then enriched.
Enrichment Protocol Summary:
An enrichment primer (containing both the amplification and sequencing primer regions) is annealed to the beads. The beads are then washed in buffer containing 2M NaCl and Tris pH 7.5 and then mixed with 1 micron biotinylated Seramag beads. This mixture is incubated at RT for three minutes on a rotator and then pelleted at 2,000 rpm in a microcentrifuge. The beads are resuspended by hand vortexing and then incubated on ice for 5 minutes. The mixture is washed on a DYNAL-MPC magnet to remove unannealed material and then NaOH is added to remove the annealed oligo. “Enriched” beads are then recovered by washing in 1× annealing buffer. 2.07 million beads were recovered from the enrichment process.
700,000 of the enriched beads were then sequenced. 414,557 of these beads had recognizable DNA sequences on them, and 206,000 of those beads had a sequence that averaged 90.5 base pairs long and were mapped to a location on the E. coli genome.
Throughout this specification, various patents, published patent applications and scientific references are cited to describe the state and content of the art. Those disclosures, in their entireties, are hereby incorporated into the present specification by reference.
|Brevet cité||Date de dépôt||Date de publication||Déposant||Titre|
|US5176203||31 juil. 1990||5 janv. 1993||Societe De Conseils De Recherches Et D'applications Scientifiques||Apparatus for repeated automatic execution of a thermal cycle for treatment of samples|
|US5225332 *||22 avr. 1988||6 juil. 1993||Massachusetts Institute Of Technology||Process for manipulation of non-aqueous surrounded microdroplets|
|US5270183||8 févr. 1991||14 déc. 1993||Beckman Research Institute Of The City Of Hope||Device and method for the automated cycling of solutions between two or more temperatures|
|US5498392||19 sept. 1994||12 mars 1996||Trustees Of The University Of Pennsylvania||Mesoscale polynucleotide amplification device and method|
|US5587128||14 nov. 1994||24 déc. 1996||The Trustees Of The University Of Pennsylvania||Mesoscale polynucleotide amplification devices|
|US5720923||31 août 1994||24 févr. 1998||The Perkin-Elmer Corporation||Nucleic acid amplification reaction apparatus|
|US5726026||14 nov. 1994||10 mars 1998||Trustees Of The University Of Pennsylvania||Mesoscale sample preparation device and systems for determination and processing of analytes|
|US5779977||26 mars 1997||14 juil. 1998||The Perkin-Elmer Corporation||Nucleic acid amplification reaction apparatus and method|
|US5827480||25 mars 1997||27 oct. 1998||The Perkin-Elmer Corporation||Nucleic acid amplification reaction apparatus|
|US5891477 *||28 mars 1997||6 avr. 1999||Biohybrid Technologies, Inc.||Non-steroidal anti-inflammatory agents inhibition of fibrotic response to an implanted device|
|US5939312||17 mai 1996||17 août 1999||Biometra Biomedizinische Analytik Gmbh||Miniaturized multi-chamber thermocycler|
|US5989892||13 juin 1996||23 nov. 1999||Tonen Corporation||Microorganisms, demulsifiers and processes for breaking an emulsion|
|US6023540||14 mars 1997||8 févr. 2000||Trustees Of Tufts College||Fiber optic sensor with encoded microspheres|
|US6033880||23 févr. 1998||7 mars 2000||The Perkin-Elmer Corporation||Nucleic acid amplification reaction apparatus and method|
|US6045676||22 août 1997||4 avr. 2000||The Board Of Regents Of The University Of California||Electrochemical detector integrated on microfabricated capilliary electrophoresis chips|
|US6132580||28 sept. 1995||17 oct. 2000||The Regents Of The University Of California||Miniature reaction chamber and devices incorporating same|
|US6143152||7 nov. 1997||7 nov. 2000||The Regents Of The University Of California||Microfabricated capillary array electrophoresis device and method|
|US6174675||27 août 1998||16 janv. 2001||Caliper Technologies Corp.||Electrical current for controlling fluid parameters in microchannels|
|US6184012||25 avr. 1997||6 févr. 2001||Medical Research Council||Isolation of enzymes|
|US6184029||27 janv. 1999||6 févr. 2001||Trustees Of The University Of Pennsylvania||Mesoscale sample preparation device and systems for determination and processing of analytes|
|US6207031||14 sept. 1998||27 mars 2001||Whitehead Institute For Biomedical Research||Methods and apparatus for processing a sample of biomolecular analyte using a microfabricated device|
|US6258858||1 mars 1999||10 juil. 2001||Japan As Represented By Director Of National Food Research Institute, Ministry Of Agriculture, Forestry And Fisheries||Cross-flow microchannel apparatus and method of producing or separating emulsions making use thereof|
|US6261431||28 déc. 1998||17 juil. 2001||Affymetrix, Inc.||Process for microfabrication of an integrated PCR-CE device and products produced by the same|
|US6266459||29 nov. 1999||24 juil. 2001||Trustees Of Tufts College||Fiber optic sensor with encoded microspheres|
|US6284525||29 août 2000||4 sept. 2001||Affymetrix, Inc.||Miniature reaction chamber and devices incorporating same|
|US6303309||21 juin 1999||16 oct. 2001||Sequenom, Inc.||Method for dissociating biotin complexes|
|US6303343||6 avr. 1999||16 oct. 2001||Caliper Technologies Corp.||Inefficient fast PCR|
|US6310354||13 juil. 1999||30 oct. 2001||Erkki Soini||Method and a device for monitoring nucleic acid amplification reactions|
|US6361671||11 janv. 1999||26 mars 2002||The Regents Of The University Of California||Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels|
|US6436355||20 nov. 1997||20 août 2002||Her Majesty The Queen In Right Of Canada, As Represented By The Secretary Of State For Defence||Electrically conducting polymer reaction vessels|
|US6489103||16 déc. 1999||3 déc. 2002||Medical Research Council||In vitro sorting method|
|US6551841||27 janv. 1999||22 avr. 2003||The Trustees Of The University Of Pennsylvania||Device and method for the detection of an analyte utilizing mesoscale flow systems|
|US6586233||9 mars 2001||1 juil. 2003||The Regents Of The University Of California||Convectively driven PCR thermal-cycling|
|US6602473||7 avr. 2000||5 août 2003||The Regents Of The University Of California||Temperature control apparatus|
|US6613560||11 févr. 2000||2 sept. 2003||Agilent Technologies, Inc.||PCR microreactor for amplifying DNA using microquantities of sample fluid|
|US6635226||11 févr. 2000||21 oct. 2003||Agilent Technologies, Inc.||Microanalytical device and use thereof for conducting chemical processes|
|US6660517||8 nov. 1996||9 déc. 2003||Trustees Of The University Of Pennsylvania||Mesoscale polynucleotide amplification devices|
|US6887664||5 juin 2001||3 mai 2005||Applera Corporation||Asynchronous primed PCR|
|US6953676||15 déc. 1998||11 oct. 2005||Trustees Of The University Of Pennsylvania||Mesoscale polynucleotide amplification device and method|
|US7005292||22 août 2003||28 févr. 2006||The Trustees Of The University Of Pennsylvania||Device and method for the detection of an analyte utilizing mesoscale flow systems|
|US7018830||21 janv. 2003||28 mars 2006||The Trustees Of The University Of Pennsylvania||Device and method for the detection of an analyte utilizing mesoscale flow systems|
|US20010020588||23 janv. 2001||13 sept. 2001||Whitehead Institute For Biomedical Research||Methods and apparatus for processing a sample of biomolecular analyte using a microfabricated device|
|US20010036632||19 déc. 2000||1 nov. 2001||Zailin Yu||Methods for amplifying and detecting multiple polynucleotides on a solid phase support|
|US20020001675||5 avr. 1999||3 janv. 2002||Thomas C. Tisone||Method for dispensing reagent onto substrate|
|US20020068357||9 août 2001||6 juin 2002||Mathies Richard A.||Miniaturized integrated nucleic acid processing and analysis device and method|
|US20020119459 *||29 juin 2001||29 août 2002||Andrew Griffiths||Optical sorting method|
|US20020168279||26 févr. 2002||14 nov. 2002||Shinya Yamamoto||Shaft seal structure of vacuum pumps|
|US20020172980||28 févr. 2002||21 nov. 2002||Phan Brigitte Chau||Methods for decreasing non-specific binding of beads in dual bead assays including related optical biodiscs and disc drive systems|
|US20030207266||5 juin 2001||6 nov. 2003||Caifu Chen||Asynchronous primed PCR|
|US20030214994||13 mai 2003||20 nov. 2003||Eppendorf Ag||Thermocycler with a temperature control block driven in cycles|
|US20040053254||17 déc. 2002||18 mars 2004||Wangh Lawrence J.||Late-pcr|
|US20050079510||28 janv. 2004||14 avr. 2005||Jan Berka||Bead emulsion nucleic acid amplification|
|US20050130173||28 janv. 2004||16 juin 2005||Leamon John H.||Methods of amplifying and sequencing nucleic acids|
|US20050202489||3 mars 2005||15 sept. 2005||Cho Yoon-Kyoung||Method and apparatus for amplifying nucleic acids|
|US20060163385||7 oct. 2005||27 juil. 2006||Link Darren R||Formation and control of fluidic species|
|US20070003442||23 févr. 2006||4 janv. 2007||President And Fellows Of Harvard College||Electronic control of fluidic species|
|EP1522581A2||6 janv. 2000||13 avr. 2005||Medical Research Council||Optical sortingmethod|
|EP1522582A2||6 janv. 2000||13 avr. 2005||Medical Research Council||Optical sorting method|
|WO1989010566A1 *||21 avr. 1989||2 nov. 1989||Massachusetts Institute Of Technology||Process for forming and using microdroplets|
|WO1997040141A2||25 avr. 1997||30 oct. 1997||Medical Research Council||Isolation of enzymes|
|WO1999002671A1||29 juin 1998||21 janv. 1999||Medical Research Council||In vitro sorting method|
|WO2000040712A1||6 janv. 2000||13 juil. 2000||Medical Research Council||Optical sorting method|
|WO2001018244A2||8 sept. 2000||15 mars 2001||Medical Research Council||Selection system|
|WO2002022869A2||13 sept. 2001||21 mars 2002||Medical Research Council||Directed evolution method|
|WO2002103011A2||18 juin 2002||27 déc. 2002||Medical Research Council||Selective gene amplification|
|WO2002103363A2||18 juin 2002||27 déc. 2002||Medical Research Council||Selection by avidity capture|
|WO2003044187A2||18 nov. 2002||30 mai 2003||Medical Research Council||Emulsion compositions|
|WO2004069849A2||28 janv. 2004||19 août 2004||454 Corporation||Bead emulsion nucleic acid amplification|
|WO2004083443A1||19 déc. 2003||30 sept. 2004||Caliper Life Sciences, Inc.||Single molecule amplification and detection of dna|
|1||Andreadis et al. (2000), Nucleic Acids Research, 28:i-viii.|
|2||Bauer: "Advances in Cell Separation: Recent Developments in Counterflow Centrifugal Elutriation and Continuous Flow Cell Separation", Journal of Chromatography B. 722 (1999) 55-69.|
|3||Brody et al "A Self-Assembled Microlensing Rotational Probe", Jan. 4, 1999, vol. 74, No. 1, pp. 144-146, 1999 American Institute of Physics.|
|4||*||Bruckner-Lea et al., Renewable microcolumns for automated DNA purification and flow-through amplification: from sediment samples through polymerase chain reaction,Analytica Chimica Acta 21616 (2001) 1-12.|
|5||*||Bruno et al., Development of an Immunomagnetic Assay System for Rapid Detection of Bacteria and Leukocytes in Body Fluids, Journal of Molecular Recognition, vol. 9.474479 (1996).|
|6||Chiou J et al.: "A Closed-Cycle Capillary Polymerase Chain Reaction Machine" Analytical Chemistry, American Chemical Society, Columbus, US, vol. 73, No. 9, May 1, 2001, pp. 2018-2021, XP-001071442.|
|7||Curcio et al: Continuous Segmented-Flow Polymerase Chain Reaction for High-Throughput Miniaturized DNA Amplification, Analytical Chemistry, vol. 75, No. 1, Jan. 1, 2003.|
|8||Dressman Devin et al: "Transforming Single DNA Molecules Into Fluroescent Magnetic Particles for Detection and Enumeration of Genetic Variation." Proceedings of the National Academy of Sciences of the United States of America. Jul. 22, 2003, vol. 100, No. 15, pp. 8817-8822, XP-002327377.|
|9||E.T. Lagally et al "Single-Molecule DNA Amplification and Analysis in an Integrated Microfluidic Device", Analytical Chemistry 2001, vol. 73, No. 3, Feb. 1, 2001, pp. 565-570.|
|10||*||Fan et al., Dynamic DNA Hybridization on a Chip Using Paramagnetic Beads,Anal. Chem. 1999, 71, 4851-4859.|
|11||Ghadessy et al "Directed Evolution of Polymerase Function by Compartmentalized Self-Replication", Apr. 10, 2001, pp. 4552-4557.|
|12||Griffiths et al: Directed Evolution of an Extremely Fast Phosphotriesterase by In Vitro Compartmentalisation, The EMBO Journal, vol. 22, No. 1 pp. 24-35, 2003.|
|13||*||Katsura et al., Indirect mincromanipulation of single molecules in water-in-oil emulsion, Electrophoresis 2001, 22, 289-293.|
|14||Katsura Shinji et al: "Indirect Micromanipulation of Single Molecules in Water-in-Oil Emulsion" Electrophoresis, vol. 22, No. 2, Jan. 2002, pp. 289-293, XP-002327329.|
|15||Kawakatsu Takahiro et al: "Regular-Sized Cell Creation in Microchannel Emulsification by Visual Microprocessing Method" Journal of the American Oil Chemists' Society, vol. 74, No. 3, 1997, pp. 317-321, XP-001206295.|
|16||*||Kopp et al., Chemical Amplification: Continuous-Flow PCR on a Chip, Science, vol. 280, May 15, 1998, pp. 1046-1048.|
|17||Kopp et al: "Chemical Amplification: Continuous Flow PCR on a Chip", Science, American Associate for the Advancement of Science, US, vol. 280, May 15, 1998, pp. 1046-1048, XP-002107956.|
|18||Lund V et al: "Assesment of Methods for Covalent Binding of Nucleic Acids to Magnetic Beads, Dynabeadstm, and the Characteristics Fo the Bound Nucleic Acids in Hybridization Reactions" Nucleic Acids Research, Oxford University Press, Surrey, GB, vol. 16, No. 22, 1988, XP-000195548.|
|19||Lundeberg J et al: "Solid-Phase Technology: Magnetic Beads to Improve Nucleic Acid Detection and Analysis" Biotechnology Annual Review, vol. 1, No. 4, 1995, pp. 373-401, XP-008044959.|
|20||Nakano Hideo et al: "High Speed Polymerase Chain Reaction in Constant Flow", Bioscience Biotechnology and Biochemistry, vol. 58, No. 2, 1994, pp. 349-352, XP-009047283.|
|21||Nakano Michihiko et al: "Single-Molecule PCR Using Water-in-Oil Emulsion.", Journal of Biotechnology, vol. 102, No. 2, Apr. 24, 2003, pp. 117-124, XP-002327328.|
|22||*||Nisisako et al., Rapid Preparation of Monodispersed Droplets With Confluent Laminar Flows, 03-7744-3/03/$17.08, 2003, IEEE, pp. 331-334.|
|23||*||Obeid et al., Microfabricated Device for DNA and RNA Amplification by Continuous-Flow Polymerase Chain Reaction and Reverse Transcription-Polymerase Chain Reaction with Cycle Number Selection, Anal. Chem. 2003, 75; 288-295.|
|24||Park N et al: "Cylindrical Compact Thermal-Cycling Device for Continuous-Flow Polymerase Chain Reaction" Analytical Chemistry, American Chemical Society. Columbus, US, vol. 75, No. 21, Nov. 1, 2003, pp. 6029-6033, XP-001047342.|
|25||Russom Aman et al: "Single-Nucleotide Polymorphism Analysis by Allele-Specific Extension of Fluorescently Labeled Nucleotides in a Microfluidic Flow-Through Device." Electrophoeresis, vol. 24, No. 1, Jan. 2003, pp. 158-161, XP-002327386.|
|26||Ruzicka (2000), Analyst, 125:1053-1060.|
|27||*||Schneegass et al., Flow-through polymerase chain reactions in chip thermocyclers, Reviews in Molecular Biotechnology 82 (2001) 101-121.|
|28||Schneegass I et al: "Miniaturized Flow-Through PCR With Different Tmeplate Types in a Silicon Chip Thermocycler" Lab on a Chip, Royal Society of Chemistry, Cambridge, GB, vol. 1, No. 1, Sep. 2001, pp. 42-49, XP-009030439.|
|29||Sepp et al: "Microbead Display by In Vitro Compartmentalisation: Selection for Binding Using Flow Cytometry", FEBS Letters 532 (2002) pp. 455-458.|
|30||Strizhkov et al. (2000), BioTechniques, 29:844-857.|
|31||Tawfik & Griffiths (1998). Nat Biotechnol 16: 652-656.|
|Brevet citant||Date de dépôt||Date de publication||Déposant||Titre|
|US8530158||10 mai 2011||10 sept. 2013||Life Technologies Corporation||System and method for processing a biological sample|
|US8552771||22 avr. 2013||8 oct. 2013||Life Technologies Corporation||System for reducing noise in a chemical sensor array|
|US8609339 *||10 sept. 2010||17 déc. 2013||454 Life Sciences Corporation||System and method for emulsion breaking and recovery of biological elements|
|US8692298||7 mai 2013||8 avr. 2014||Life Technologies Corporation||Chemical sensor array having multiple sensors per well|
|US8731847||30 juin 2011||20 mai 2014||Life Technologies Corporation||Array configuration and readout scheme|
|US8741680||22 mars 2013||3 juin 2014||Life Technologies Corporation||Two-transistor pixel array|
|US8742471||21 mars 2013||3 juin 2014||Life Technologies Corporation||Chemical sensor array with leakage compensation circuit|
|US8742472||19 juin 2013||3 juin 2014||Life Technologies Corporation||Chemically sensitive sensors with sample and hold capacitors|
|US8766328||20 juin 2013||1 juil. 2014||Life Technologies Corporation||Chemically-sensitive sample and hold sensors|
|US8786331||13 mars 2013||22 juil. 2014||Life Technologies Corporation||System for reducing noise in a chemical sensor array|
|US8841217||13 mars 2013||23 sept. 2014||Life Technologies Corporation||Chemical sensor with protruded sensor surface|
|US8890216||4 juin 2013||18 nov. 2014||Life Technologies Corporation||Methods and apparatus for measuring analytes using large scale FET arrays|
|US8912005||17 juil. 2014||16 déc. 2014||Life Technologies Corporation||Method and system for delta double sampling|
|US8912580||12 mars 2014||16 déc. 2014||Life Technologies Corporation||Active chemically-sensitive sensors with in-sensor current sources|
|US8962366||28 janv. 2013||24 févr. 2015||Life Technologies Corporation||Self-aligned well structures for low-noise chemical sensors|
|US8963216||13 mars 2013||24 févr. 2015||Life Technologies Corporation||Chemical sensor with sidewall spacer sensor surface|
|US8983783||11 avr. 2013||17 mars 2015||Life Technologies Corporation||Chemical detection device having multiple flow channels|
|US9023189||19 avr. 2013||5 mai 2015||Life Technologies Corporation||High density sensor array without wells|
|US9110015||12 déc. 2014||18 août 2015||Life Technologies Corporation||Method and system for delta double sampling|
|US9116117||5 mars 2014||25 août 2015||Life Technologies Corporation||Chemical sensor with sidewall sensor surface|
|US9128044||5 mars 2014||8 sept. 2015||Life Technologies Corporation||Chemical sensors with consistent sensor surface areas|
|US9134269||11 mars 2013||15 sept. 2015||Life Technologies Corporation||Methods and apparatus for measuring analytes using large scale FET arrays|
|US9174217||7 sept. 2012||3 nov. 2015||Samsung Electronics Co., Ltd.||Device and method of amplifying nucleic acids by using oil layer comprising exothermal particles|
|US9194000||22 oct. 2014||24 nov. 2015||Life Technologies Corporation||Methods and apparatus for measuring analytes using large scale FET arrays|
|US9239313||4 sept. 2014||19 janv. 2016||Life Technologies Corporation||Ion-sensing charge-accumulation circuits and methods|
|US9270264||17 juil. 2014||23 févr. 2016||Life Technologies Corporation||System for reducing noise in a chemical sensor array|
|US9403141||16 oct. 2015||2 août 2016||Twist Bioscience Corporation||De novo synthesized gene libraries|
|US9404920||9 janv. 2014||2 août 2016||Life Technologies Corporation||Methods and apparatus for detecting molecular interactions using FET arrays|
|US9409139||5 août 2014||9 août 2016||Twist Bioscience Corporation||De novo synthesized gene libraries|
|US9555388||20 juin 2016||31 janv. 2017||Twist Bioscience Corporation||De novo synthesized gene libraries|
|US9624519||22 août 2014||18 avr. 2017||454 Life Sciences Corporation||System and method for nucleic acid amplification|
|US9677067||13 mai 2016||13 juin 2017||Twist Bioscience Corporation||Compositions and methods for synthetic gene assembly|
|US9808806||25 août 2014||7 nov. 2017||454 Life Sciences Corporation||System and method for automated nucleic acid amplification|
|US20110087016 *||10 sept. 2010||14 avr. 2011||454 Life Sciences Corporation||System and Method for Emulsion Breaking and Recovery of Biological Elements|
|USRE43365||27 sept. 2010||8 mai 2012||Lawrence Livermore National Security, Llc||Apparatus for chemical amplification based on fluid partitioning in an immiscible liquid|
|USRE45539||30 mars 2012||2 juin 2015||Lawrence Livermore National Security, Llc||Method for chemical amplification based on fluid partitioning in an immiscible liquid|
|USRE46322||30 avr. 2015||28 févr. 2017||Lawrence Livermore National Security, Llc||Method for chemical amplification based on fluid partitioning in an immiscible liquid|
|EP2840148A1||14 août 2014||25 févr. 2015||F. Hoffmann-La Roche AG||System and method for nucleic acid amplification|
|EP2848698A1||14 août 2014||18 mars 2015||F. Hoffmann-La Roche AG||System and method for automated nucleic acid amplification|
|WO2011135041A1||28 avr. 2011||3 nov. 2011||Roche Diagnostics Gmbh||System and method for purification and use of inorganic pyrophosphatase from aquifex aeolicus|
|WO2011144682A2||19 mai 2011||24 nov. 2011||Roche Diagnostics Gmbh||System and method for tailoring nucleotide concentration to enzymatic efficiencies in dna sequencing technologies|
|WO2012041857A1||27 sept. 2011||5 avr. 2012||Roche Diagnostics Gmbh||System and method for producing functionally distinct nucleic acid library ends through use of deoxyinosine|
|WO2012045820A1||6 oct. 2011||12 avr. 2012||Roche Diagnostics Gmbh||System and method for detection of hiv-1 clades and recombinants of the reverse transcriptase and protease regions|
|WO2012130681A1||21 mars 2012||4 oct. 2012||Roche Diagnostics Gmbh||System and method for detection of hiv integrase variants|
|WO2013124390A1||22 févr. 2013||29 août 2013||Roche Diagnostics Gmbh||System and method for generation and use of compact clonally amplified products|
|WO2013171303A2||16 mai 2013||21 nov. 2013||Roche Diagnostics Gmbh||System and method for generation and use of optimal nucleotide flow orders|
|Classification aux États-Unis||435/6.16, 435/91.2, 239/88|
|Classification internationale||C12P19/34, B01L7/00, C12Q1/68, F02M47/02, B01F13/00, C40B40/06, B01L3/00, B01J19/00, B01F3/08, C40B60/14, B01F5/04, G01N35/10|
|Classification coopérative||G01N35/1095, B01J2219/00495, B01L7/525, B01L2300/1844, B01J2219/00722, B01J2219/005, B01L2300/1827, B01F5/0413, C40B60/14, B01L2400/0439, C40B40/06, B01L2400/0487, B01L2200/0673, B01F13/0059, B01F5/0471, C12Q1/686, B01F3/0807, B01J2219/00664, B01L3/5027|
|Classification européenne||C12Q1/68D4, G01N35/10V, B01F5/04C14, B01F13/00M, B01F3/08C, B01L7/525|
|24 juin 2005||AS||Assignment|
Owner name: 454 CORPORATION, CONNECTICUT
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NOBILE, JOHN R.;LEE, WILLIAM L.;LEAMON, JOHN H.;REEL/FRAME:016717/0969;SIGNING DATES FROM 20050512 TO 20050523
Owner name: 454 CORPORATION, CONNECTICUT
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NOBILE, JOHN R.;LEE, WILLIAM L.;LEAMON, JOHN H.;SIGNING DATES FROM 20050512 TO 20050523;REEL/FRAME:016717/0969
|7 mars 2011||AS||Assignment|
Owner name: 454 LIFE SCIENCES CORPORATION, CONNECTICUT
Free format text: CHANGE OF NAME;ASSIGNOR:454 CORPORATION;REEL/FRAME:025913/0157
Effective date: 20030917
|28 nov. 2014||REMI||Maintenance fee reminder mailed|
|19 avr. 2015||LAPS||Lapse for failure to pay maintenance fees|
|9 juin 2015||FP||Expired due to failure to pay maintenance fee|
Effective date: 20150419

domain: chemistry


This certainly has been a complicated week to be an American Jew.
We have completed Rosh Hashanah and Yom Kippur, in all their white-draped abstraction, and turned to the physicality of Sukkot, with its harvest symbols and waving branches and the delicate yellow scent of the etrog, with all the meals, often heavy and rich to ward off the October chill. Beyond that, we turn to the joyous physicality of Simchat Torah, with its dancing and joy, and its knowledge that this is it, the last of the holidays, and the turn toward winter.
That’s the Jewish part.
In the outside world, there seems to be nothing but devastation. Puerto Rico — a place many of us have been to and can visualize in its prelapsarian state — is demolished. It makes the destruction of Houston and Florida, which was bad, seem to be not such a big deal, at least by comparison. (I know, it’s easy to say that from the safety of the Northeast. Superstorm Sandy was a big problem for us, but not nearly as huge a problem as Maria, and anyway it was five years ago.) As our president has reminded us, it is hard to help Puerto Rico because, as he put it, it is “surrounded by water, big water, ocean water.” Who knew?
And then the impossible-to-assimilate evil of Las Vegas, of a man so unimaginably devoid of humanity that he was able to shoot through a 32nd-floor window to the happy unsuspecting crowd below as if he were playing a video game.
So far we have no idea why he did what he did. All we know is that there is no obvious reason. We might never be able to understand his diseased thinking well enough to understand it, and we might not want to be able to enter that putrid stinking brain long enough to try.
But we do know that he had an arsenal. News reports tell us that he had 23 guns with him in the hotel room; 12 could fire automatically. He had 19 other weapons at home, as well as ammunition and explosives. His truck held “ammonium nitrate, a chemical that can be used in bombmaking,” the Washington Post said. And then there’s the bump stock, something new that we had no need ever to have heard about, a grotesque but cheap way of turning a semiautomatic gun into a virtual automatic. (Guns outfitted with bump stocks apparently cannot aim well – but who needs to aim at a crowd of 22,000? And they overheat quickly – so why not have 12 of them, and move from gun to gun?)
Even if we do not and cannot know why the murderer wanted this devil’s cache, we do know why he was able to have it. We do not know if all of it was legal, but we do know that the gun lobby has made sure that guns are never far from the hot, imprudent hands of almost anyone who wants them.
It’s the Second Amendment, we’re told.
This is the Second Amendment:
A well regulated Militia, being necessary to the security of a free State, the right of the people to keep and bear Arms, shall not be infringed.”
I understand that as time passes, the kernel of a law often gains accretions, ways of understanding it, and putting it into practice. Halacha, Jewish law, works that way. Over the course of millennia, the original Torah prescriptions, against, say, boiling a kid in its mother’s milk have become the elaborate laws of kashrut that govern what and when and how observant Jews eat. The connection between the Torah law and the dinner plate may not always be clear, but it always is traceable.
So what about the Second Amendment? It is newer by millennia than Torah law, but it seems to bear about as much resemblance to the Las Vegas mass murderer’s cache as the Torah law does to halacha.
I am not a lawyer. I don’t know how legal reasoning works. But I do understand close reading of text, and I do not see what a well regulated militia has to do with that monstrous arsenal.
It is time to rethink our blind allegiance to the accretions on the Second Amendment and to restore our clear-sighted allegiance to the Declaration of Independence’s promise of life, liberty, and the pursuit of happiness — none of which you can have if you are mown down by an assault rifle — by stripping those accretions away.
The NRA should not have the final word. That should belong to we, the people.


text:
OWINGS MILLS, Md. — The Ravens were forced to cancel practice by the NFL one day prior to their Week 12 game against the Steelers because of ongoing concerns with COVID-19, according to a report by ESPN.
This means Baltimore has not practiced in 10 days because of the number of players that have tested positive for the virus.
After wide receiver Willie Snead reportedly tested positive late Sunday night, the Ravens now have 23 players on the COVID-19 list, including 11 starters, seven players who made the Pro Bowl last season, and reigning NFL MVP Lamar Jackson.
The game against Pittsburgh was originally scheduled for Thanksgiving night but has been pushed back until Tuesday, Dec. 1 because of the outbreak among the Ravens players.
Baltimore is still finalizing plans for when to travel to Pittsburgh, which could get up to three inches of snow on Tuesday, further complicating travel.
If the game is played, the Ravens will be shorthanded on both sides of the ball.
The Ravens currently have six offensive linemen available for the game —tackle, Orlando Brown Jr., guard Bradley Bozeman, rookie center Trystan Colon-Castillo, rookie guards Ben Bredeson and Tyre Phillips and second-year guard Ben Powers.
Baltimore is dealing with the same challenges on the defensive line.
Baltimore has a competent backup quarterback in Robert Griffin III, but he will have to play behind a makeshift offensive line against a fierce Steelers pass rush that leads the NFL with 38 sacks.
If the Ravens have any more positive tests the day prior to the game, the NFL could postpone the matchup. The NFL could create a Week 18 to play this game and others affected by the virus,
At this point, it's all about survival.
The Ravens COVID list includes:
QB Lamar Jackson
QB Trace McSorley
RB Mark Ingram II
RB J.K. Dobbins
WR Willie Snead
TE Mark Andrews
C Patrick Mekari
C Matt Skura
FB Patrick Ricard
OL D.J. Fluker
T Will Holden
DT Brandon Williams
DE Calais Campbell
DE Jihad Ward
DT Justin Madubuike
OLB Jaylon Ferguson
OLB Pernell McPhee
OLB Matt Judon
DT Broderick Washington
CB Iman Marshall (IR)
CB Tavon Young (IR)
CB Khalil Dorsey (IR)
LS Morgan Cox

domain: news


The Fifa World Cup 2018 has been full of surprises. In order to keep Bangladeshi viewers up to date with the ups and downs of this capricious tournament, popular TV host Maria Nur has been hosting a pre-match talk show “Football Paa-Goal with Maria.” She has often been termed the top sports host in the country, even though she’s not from a sports fraternity. In an interview with Dhaka Tribune Showtime’s Sadia Khalid, model and TV host Maria Nur shares her thoughts on this World Cup and her show.
How long have you been hosting sports shows?
It has been four years.
In Bangladesh, we often have sports show hosts from non-sports backgrounds, unlike Harsha Bhogle, Sunil Gavaskar or other cricketers hosting cricket shows internationally. Do you think being from a different background affects the quality of the show’s content?
There are hosts from non-sports backgrounds internationally as well. Sports journalists are also often not from sports backgrounds. If I talk about myself, personally, I don’t only host sports shows. In sports shows, my role is that of a presenter, a moderator. My job is to ask questions to the analysts or to other guests at my show. They’ll answer. It is not my job to analyze. In that case, coming from a sports or non-sports background doesn’t matter. What matters is if the person is a sports fan, a sports lover. If the quality of the show would drop, then viewers wouldn’t want Maria to host sports shows. The slogan “Cricket means Maria” wouldn’t be created in Bangladesh. When I’m doing a football show, it wouldn’t also be appreciated. It’s all about an individual’s performance, how she presents herself.
Recently the Bangladesh women’s cricket team won the Asia Cup, but we still don’t see many female analysts on sports shows. Why do you think that is?
I’ve had female sports journalists on my show. There are two types of guests on my show, experts from the sports arena and celebrities. In the last semi-final match (of the Fifa World Cup 2018), I had the honourable information minister, Hasanul Haq Inu, as my guest. I’ve had female guests on my show in both categories.
In Bangladesh, gender discrimination is deeply rooted. We think girls don’t understand sports, or girls can’t talk about sports. A lot of time has passed, but we still haven’t been able to come out of that frame of mind. When we appear on the TV screen as sports hosts, we have to face a lot of criticism. I’ve also had to deal with negative comments. A lot of people say that girls host sports shows to attract viewers. But I’m working hard to make my shows stand out. That’s why people are now saying: “Cricket means Maria. Football also means Maria.” We haven’t been able to free ourselves from the conservativeness and gender discrimination.
Would you like to share any memorable comments from your World Cup show this year?
That’s a toughie. There were many notable comments. The comment we received the most this time around is: “This World Cup is full of surprises.” There’s nothing called a hot favourite here. There are no small teams among these 32, who qualified for this World Cup. The dark horses have put the so-called big teams to the test. That has made this World Cup different from others. I think that’s why people are still watching the matches even after Argentina and Brazil went home.
Which is your favourite sport- cricket or football?
Personally, because of the Bangladesh team, cricket is my favourite sport. When we grew up, the golden age of football for Bangladesh had already passed. For that reason, we watched more cricket than football. But I love watching football as well.
How often do you get to watch the games?
If Bangladesh is playing, I watch the matches no matter how busy I am. Now that Bangladesh is playing a cricket series during the football World Cup, I’m missing those matches.
Who has been your favourite player in this world cup?
There are so many. Romelu Lukaku from Belgium, Luka Modrić from Croatia. Messi is my all time favourite. Then there is Cristiano Ronaldo, Neymar, Mbappe. Danijel Subašić from Croatia was my favourite goalkeeper. I was a fan of the dark horses throughout the World Cup.
Who are you supporting in the final match?
I can’t say that before that final show ends. I have to stay neutral as a host.


Thursday, January 9, 2014
CES: TrackingPoint's computer-guided smart rifle can make every hunter a sharpshooter
PCWorld - LAS VEGAS—The TrackingPoint AR Smart Rifle is powerful and deadly—even in the “wrong” hands.
Geared toward sport hunters, this Precision Guided Firearm is a closed-loop system, not just a high-tech scope. The system includes a custom rifle manufactured by Surgeon, a Networked Tracking Scope by TrackingPoint, and a guided trigger, all of which work in tandem to ensure a more accurate shot from as much as 500 yards away.
Basically, this rifle helps less-experienced hunters become skilled marksmen in a second, all thanks to technology.
Here’s how it works: The TrackingPoint AR Smart Rifle features TrackingPoint’s Tag Track Xact system, which lets hunters tag multiple targets from far away. Once a target has been tagged through the TTX system, all the hunter has to do is pull the trigger—the system does the rest. It lines up the shot, electronically controls for external variables, and only then does it actual fire the shot, once everything is in place. Read More


Make musical sounds with the voice, especially words with a set tune.
She loves to sing every chance she gets.
Your information is 100% private & never shared.See Past Email Examples
Copyright © 2010 –
HomeSpeechHome.com | All Rights Reserved


text:
Have you ever felt gunky, gassy, and lethargic after a meal? If your day of eating usually includes takeout, waffle fries and Caesar dressing, you probably know what I’m talking about. That slight tug in your belly after you’ve finished off a Mcflurry, that deep desire to fall into a sleep coma after a buttery grilled cheese sandwich…. You know what I mean.
The meal we’re talking about today will provide you with nourishment, satisfaction and deliciousness. Oh, and for anyone out there who is constantly in a time crunch, this recipe is quick and easy to make. So you have no excuse not to prep this lil’ meal. And although this recipe is a “Buddha Bowl,” I promise you won’t look like Buddha after licking the bowl clean.
This meal checks off most of the items on your daily needs list — Superfood? Check. Veggies? Check. Protein? Check. Healthy fats, grains and greens? Check, check, and — you guessed it — check.
Here’s what you’ll need:
1 cup spinach
1/2 cup cooked quinoa
1/2 cup cooked chickpeas
1/4 cup chopped purple cabbage, lightly steamed
1 cup veggies of your choice
*I mini red peppers, buttercup squash, and spaghetti squash
1/2 avocado, mashed
1 tsp olive oil
1 tsp lime juice
Pinch garlic powder
Salt + pepper
What You Do:
Prepare quinoa and chickpeas according to package directions.
Steam cabbage in 1 inch water on stovetop.
Lightly season your veggies with olive oil and salt and pepper, then roast in the oven 400F.
Layer all base ingredients into bowl (make it look pretty!)
To make the dressing, combine all ingredients in a food processor or blender. You can also mash the avocado by hand, but the dressing won’t come out as smooth and creamy.
Drizzle dressing over your bowl and voila!
I hope you enjoy this recipe as much as I do. I personally love how versatile it is — you can swap out the quinoa for brown rice, the chickpeas for black beans, and the spinach for kale or romaine — which prevents you from getting bored throughout the week.

domain: food


text:
On March 8, 2023 DID Executive Committee issued a letter to our New York State representatives in support of the 13th Forward, a part of the Justice Roadmap previously ratified by DID membership.
The letter began:
Dear Reps. Lee, Glick, Fall, and Heastie, and Sens. Kavanagh, Hoylman-Sigel, and Stewart-Cousins,
Downtown Independent Democrats requests our reelected and newly elected State representatives to please sponsor the 13th forward bill, which DID Membership endorsed on May 26, 2021 via the resolution in Support of the Justice Roadmap. We thank Hon. Brad Hoylman-Sigel for signing on.
13th Forward is a statewide coalition of organizers, directly impacted individuals, faith leaders, and workers’ and criminal legal reform advocates advancing the No Slavery in NY Act (S.225 Myrie/A.3412 Epstein) and the Fairness and Opportunity for Incarcerated Workers Act (bill numbers pending) to end slavery and exploitation in New York State.

domain: politics


text:
Location: Prizren, Kosovo listening to the muezzin all accros town calling to prayer at sunset. quite an atmopshere from the Prizren fortress overlooking the city... www.francoismarclay.com | Tumblr
Page not found - The Lovely Planet
Women of all races, sects, religions or nations deserve true respect and prestige. The purpose of the Women of the Lovely Planet series is to pay a special tribute to the women of the world. We honor the roles of the mother, the sister, the aunt, the wife and the daughter as they toil the most for their families.
Patriarchate of Pec, Kosovo
UNESCO protected spiritual seat of the Old Serbian Kingdom and Empire, this complex contains from 4 churches built between 13th and 14th centuries. After the empire collapsed, muslims have started to settle in the area, while Christians began leaving Kosovo, fearing revenge and repression. The peak of the repression were the last decades of the 16th centuries, when this Patriarchate has been abolshed as well as freedom of religion in Serbia, resulting in the Serbian rebellion of 1595. That…

domain: geography


Leicester City defender Wesley Fofana is reportedly in talks over a new contract.
The 21-year-old is said to have been in talks for "several weeks" and could extend until the summer of 2027.
With that said, there is no rush for Leicester to agree a new deal as his current deal expires at the end of the 2024-25 season.
Leicester will be keen to tie their star defender down, though, amid reported transfer interest from the likes of Chelsea and Real Madrid.
They also say Fofana could make his playing return in that game, although it seems likely he will start from the bench at best.
Fofana has been back in full training with his teammates since February 14 and will be itching to get back out onto the pitch in a competitive match.
Getting Fofana tied down to a longer deal would be a real show of intent and ambition for Leicester, who are unlikely to be playing European football next season - unless they win the Europa Conference League.
He would be an important focal point for the team heading into next season and beyond, with a summer transfer overhaul on the horizon in the coming months.
Fofana, Kiernan Dewsbury-Hall and Patson Daka are an exciting young core to build a fresh team around for future success in the Premier League and, hopefully, Europe.


Doreen Lawrence has changed attitudes and policing in the UK, Labour leader Ed Milband said today after it was announced that she would be made a Labour peer.
Mr Miliband said: "Over the past 20 years, Doreen Lawrence has had a profound impact on Britain and I am delighted that she will become a Labour member of the House of Lords.
"Since the horrific racist murder of her son, Doreen has shown incredible strength and courage as she sought, and continues to seek, justice for Stephen.
"She has changed attitudes to policing and racism in this country and I have no doubt that her strength and determination will be a huge asset to the House of Lords in the coming years."
More top news
Services on major rail routes between London and south west England have been severely disrupted after a person was hit by a train.
The business executive, whose ruthless interviewing technique has won him many fans, is replacing Nick Hewer, who quit after a decade.
Recent opinion polls show Labour within striking distance of capturing the trophy seat of Finchley and Golders Green.


text:
Lademadera powder-coated aluminium
Agora Liso by Tuvatextil
Agora Panama by Tuvatextil
Amelia by Reviva
Bora Bora by Crevin
Bora bora 99
Bora bora 08
Bora bora 05
Bora bora 54
Bora bora 50
Bora bora 59
Bora bora 53
Dolce by Crevin
Azul grisáceo 45
Azul oscuro 42
Gris oscuro 53
Duo by Crevin
Gris claro 51
Gris oscuro 53
Gemini by Benecke Kaliko
Iris by Reviva
Libera by Dedar Milano
Natté by Sunbrella®
Patio by Kvadrat
Silvertex by Spradling®
Sling by Sunbrella®
Solids by Sunbrella®
Domino by Sunbrella® (only pillows)
Majestic by Sunbrella® (only pillows)
Strange Loves by Dedar Milano (only pillows)
Sale e Pepe
Lademadera 2 Seat sofa
“Wood has been used by humans since ancient times for the construction of houses, furniture, tools, and various objects, and it represents the material closest to man. It is a familiar material that has always fascinated us.”
Francesco Meda and David Quincoces
Purity and simplicity in forms, and care in the choice of materials characterize the LADEMADERA collection, designed by Francesco Meda and David Quincoces for GANDIABLASCO. The result is a collection of environmentally friendly outdoor furniture, whose natural finishes and clean lines make it integrate in a balanced way into any architectural space.
The LADEMADERA two-seater sofa is a designer sofa made of teak wood obtained from sustainably managed forests and a 100% recyclable powder-coated aluminum structure. In combination with the lounge chair or the two tables from the collection, it is a perfect solution for any type of environment, both indoors and outdoors.
The LADEMADERA two-seater sofa is an original design by Francesco Meda and David Quincoces for GANDIABLASCO.
Seat and backrest in polyurethane foam rubber and polyester fibre with water-repellent fabric
Polyester fibre cushion/pillow with water-repellent fabric
Removable technical upholstery for outdoor use
About Francesco Meda & David Quincoces
Francesco Meda (Milan, 1984) graduated in 2006 from the European Institute of Design in Milan. The designer gained experience in London working in Sebastian Bergne’s studio and later Ross Lovegrove’s.
In 2008 he returned to Milan and collaborated with his father Alberto Meda on brands such as Kartell and Caimi Brevetti. Meda has promoted her own art and design projects in other international firms and prestigious galleries such as Nilufar Gallery and Schoeni Art Gallery in Hong Kong. Among the applauded designs of him, stand out the bridge lamps and the 3D printed jewels that are part of the collection of the Triennale Design Museum in Milan.
Francesco Meda also has several awards. The last one was the 2019 Wallpaper Design Awards with the Woody chair for Molteni & C.
David Quincoces, born in Madrid, has lived in Milan since 2005, where he arrived to study a master’s degree in interior design at the Politecnico di Milano. Having worked with Piero Lissoni, he founded his own studio, Quincoces-Dragó, more than ten years ago.
It is currently one of the most sought-after architecture firms in Milan and works both in collaboration with design companies and in interior architecture projects.
Francesco Meda & David Quincoces

domain: design


text:
Original Posting At https://beguineagain.com/testimony-tuesday-candles/
Sometimes things just land in your lap and you say, “Thank you” and go with it! This is just such a day. We have a guest posting from Joan Leotta. Joan is an author and story performer. She arrived on my email doorstep this week and I am delighted to share her gifts.
As testimony Tuesday is dedicated to pointing towards the truth of things, I feel her gracious offering of Candles is in order. I am very grateful to her generosity! Thank you, Joan!
by Joan Leotta
que non se apaguen
tus ideas (amnesty international)
On my shirt, a
surrounded by barbed wire,
marks light’s role
relieving a world of darkness.
In my heart,
ideas flicker, grow strong
as I live the admonition
of my shirt
to make real the words
of my ideals with my pen
and daily life performance.
Writing, living these
bridges that vast canyon
prayer and practice, the
holy grace of candles
banishing the dark
and making life bearable on
an unholy earth.
Author, Story Performer
“Encouraging words through Pen and Performance”
Giulia Goes to War, Letters from Korea, A Bowl of Rice, Secrets of the Heart
historical fiction in Legacy of Honor Series
Simply a Smile – collection of Short Stories
WHOOSH! Picture book from THEAQ
You can download a mini-chapbook of her poems here.
Find out about more of her work here.

domain: artistic


5/15/2013 9:39:00 AM School honors outstanding contributors May 22
Each year, folks in the Harbor Springs School District gather to celebrate community members who are making profound contributions to the education and well-being of students. The annual GIVE celebration will take place this year on Wednesday, May 22 at 7 p.m. in the Performing Arts Center.
"It's a chance for us to say thank you, but also, a way to bring the community together to recognize just how special this place is," said superintendent Mark Tompkins. "We are so fortunate to have the dedication and care we have when it comes to our students, and this is a chance to be inspired."
This year's GIVE (Generosity, Inspiration, Volunteerism, and Excellence) recipients include Carol McKee's third grade class, Molly Ames Baker, the Harbor Springs Athletic Boosters, and Suzie Johnson and the Young Americans.
Each honoree is collectively chosen by the administrative team, Tompkins said.
Carol McKee's third grade class exemplifies Generosity, Tompkins noted, because of the tens of thousands of dollars they have raised for St. Judes in the annual Math-a-thon.
Molly Ames Baker is being honored for Inspiration. Her Learn Local initiatives have impacted many students in the district, from the SOLO (Special Outdoor Learning Opportunities) she does with middle school students to weekly activities in the elementary school.
"Molly is a perfect example of what happens when a community member has passion (and professional experience-- she and husband Josh ran Colgate's outdoor education program for over a decade) and is willing to share that with students, teachers, and administrators," Tompkins said.
He said the recipients of the Volunteer award-- the Harbor Springs Athletic Boosters-- are so dedicated that it's hard to quantify the difference the organization has made in the lives of student athletes.
"Every sporting event we have, members of the athletic boosters are there," he said. "I'm not sure you'd find a harder working bunch of volunteers anywhere."
For excellence, Tompkins said the award for Suzie Johnson and the Young Americans represents a growing partnership between the district and the performing arts organization.
"The Young Americans summer camp last year blew us all away and raised a tremendous amount of money for the arts in our district. We're so excited to see our connection to them continue to grow."
In between each recognition, event-goers will be treated to a performing arts showcase that will include the high school jazz band, and a number from the elementary school play, Seussical the Musical.


text:
A Russian startup created a human-like robot for offices and airports
Publication/Creation DateOctober 31 2019
DescriptionRussian startup Promobot built what it calls the world's first android that looks like a real person and could be coming to the workplace in the near future. With life-like movements and speech, it could perform tasks in offices, airports, banks, museums, as well as the home. The company has over ten orders for this latest innovation in artificial intelligence.
Date archivedNovember 21 2019
Last editedNovember 21 2019