WO2010009426A2 - Devices and methods for reagent delivery - Google Patents
Devices and methods for reagent delivery Download PDFInfo
- Publication number
- WO2010009426A2 WO2010009426A2 PCT/US2009/051041 US2009051041W WO2010009426A2 WO 2010009426 A2 WO2010009426 A2 WO 2010009426A2 US 2009051041 W US2009051041 W US 2009051041W WO 2010009426 A2 WO2010009426 A2 WO 2010009426A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- reagent
- reaction
- delivery device
- pcr
- sequencing
- Prior art date
Links
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/02—Burettes; Pipettes
- B01L3/0289—Apparatus for withdrawing or distributing predetermined quantities of fluid
- B01L3/0293—Apparatus for withdrawing or distributing predetermined quantities of fluid for liquids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1002—Reagent dispensers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0442—Moving fluids with specific forces or mechanical means specific forces thermal energy, e.g. vaporisation, bubble jet
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
Definitions
- the present teachings relate to devices and methods for delivering reagents during a reaction.
- the present teachings relate to devices and methods for automated reagent delivery during a reaction.
- Various biochemical and/or chemical reaction workflows require numerous stages in which controlling parameters of the reaction differ.
- differing stages of a reaction may include differing temperatures, times, addition and/or removal of supporting reagents and other substances from the reaction, and/or other parameters that may need to be altered during the reaction.
- conventional workflows involved in performing a multi-stage reaction involve various manual steps.
- a conventional polymerase chain reaction (PCR)/sequencing workflow generally includes three stages, each of which requires reagent addition. Those stages include an initial PCR stage, a cleanup stage, and a sequencing stage.
- the PCR stage involves amplification of a template polynucleotide using amplification primers and a thermo-stable DNA polymerase enzyme.
- the cleanup stage is commonly performed by adding exonuclease I and alkaline phosphatase, followed by incubation, and subsequent heat-kill to inactivate the enzymes.
- sequencing primers and reagents such as, for example, dNTPs (deoxynucleotide triphospates), ddNTPs-dyes (dideoxynucleotide triphospates-dyes), and polymerase may be added and a thermal cycle employed to perform sequencing.
- dNTPs deoxynucleotide triphospates
- ddNTPs-dyes dideoxynucleotide triphospates-dyes
- polymerase a thermal cycle employed to perform sequencing.
- FIG. 1 A standard PCR/sequencing workflow is illustrated in FIG. 1.
- Typical PCR uses an excess of amplification primers, some of which remain even upon completion of the reaction. Further, the PCR reaction products may contain excess dNTPs. Because excess amplification primers and excess dNTPs can interfere with a subsequent sequencing reaction, they are generally removed in a cleanup stage after the PCR reaction through the addition of exonuclease I and alkaline phosphatase enzymes.
- the exonuclease I and alkaline phosphatase enzymes are subject to a heat-kill step to inactivate those enzymes.
- sequencing reagents including, for example, sequencing primers, dNTPs and ddNTPs-dyes are added to the remaining mixture and sequencing is performed.
- the conventional PCR/sequencing workflow described above involves two reagent addition steps after the PCR stage: (1 ) the reagent addition to remove the excess dNTPs and amplification primers and, thereafter, (2) the reagent addition to support sequencing.
- those reagent addition steps are done manually, typically by opening the vessel (or vessels) in which the reactions are taking place and adding the reagents.
- PCR/oligonucleotide ligation assay Another example of a multi-stage reaction that involves a reagent addition stage after performing PCR is PCR/oligonucleotide ligation assay (OLA).
- OVA PCR/oligonucleotide ligation assay
- a multiplex PCR reaction (with multiple primer pairs) is performed via thermal cycling, which may include a relatively long heat-kill step to kill the DNA polymerase (for example, at a temperature of about 95 0 C for at least about 30 minutes.)
- the reaction vessel is opened and thermostable ligase, ATP (adenosine-5'-triphosphate), dye-labeled and/or dragchute labeled probes are added (e.g., via pipetting or other manual, invasive mechanism).
- the reaction vessel is then closed and several cycles of cyclic ligation are performed (e.g., involving annealing, ligating, and denaturing). Once the several cycles are completed, the reaction vessel is opened and loaded onto a capillary electrophoresis instrument. Contrary to a PCR/sequencing workflow, the PCR/OLA workflow does not require a cleanup step after PCR. Moreover, unlike some reagents that may be used in PCR/sequencing workflows, in an exemplary process, the ligase used in PCR/OLA may be thermostable and therefore the risk of denaturing it (e.g., rendering it inactive) with heat before delivery is minimized.
- Various other biochemical and/or chemical multi-stage reaction workflows also may require the addition of reagent to the reaction mixture after an initial reaction stage, such as, for example, an initial PCR stage.
- an initial reaction stage such as, for example, an initial PCR stage.
- the particular multi-stage reactions described generally above and in more detail below are exemplary and non-limiting. Those having ordinary skill in the art will recognize other assays that may require the addition of reagents at various stages of a reaction.
- the present invention may solve one or more of the above-mentioned problems. Other features and/or advantages may become apparent from the description which follows.
- the present teachings contemplate a method for delivering reagent during a thermal cycling reaction that includes placing a delivery device containing a reagent proximate to a reaction site, initiating a first stage of the reaction, wherein the first stage includes a period of an elevated temperature, and after completing the first stage of the reaction, dispensing the reagent from the delivery device to the reaction site, wherein the dispensing includes automatically actuating the delivery device to deliver the reagent.
- the present teachings contemplate a kit for sequencing nucleic acids that includes a reagent comprising a nuclease and a nuclease-resistant sequencing primer and a dispensing device holding the reagent, wherein the dispensing device is configured to automatically deliver the reagent to a reaction site.
- the present teachings contemplate a method of sequencing nucleic acids that includes positioning a delivery device holding a reagent proximate a reaction site, amplifying nucleic acids at the reaction site in a first reaction to form amplified nucleic acids, wherein the first reaction comprises thermal cycling.
- the method may include automatically actuating the delivery device to deliver the reagent to the reaction site and causing the amplified nucleic acids to react in a sequencing reaction with the reagent.
- FIG. 1 is a schematic diagram representing a conventional PCR/ sequencing workflow
- FIG. 2 is a schematic diagram representing a PCR/sequencing workflow comprising two stages and utilizing a nuclease-resistant sequencing primer according to various exemplary embodiments of the present teachings;
- FIG. 3 is a plan view of an exemplary embodiment of a reagent delivery device in accordance with the present teachings
- FIGS. 4A-4C show exemplary steps for delivering a reagent during a workflow using the reagent delivery device of FIG. 3;
- FIG. 5A is an exemplary embodiment of a dispenser element of a reagent delivery device in accordance with the present teaching
- FIG. 5B is a cross-sectional view of the dispenser element taken through line 5B-5B in FIG. 5A;
- FIG. 6 is a perspective view of an exemplary embodiment of a strip cap
- FIG. 7 is a cross-sectional view of an exemplary embodiment of a reagent delivery device in accordance with the present teachings
- FIG. 8 is a cross-sectional view of the reagent delivery device of FIG. 7 in cooperation with a reaction chamber to deliver reagent thereto;
- FIGS. 9A and 9B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIGS. 10A and 10B are cross-sectional views of another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIGS. 11 A and 1 1 B are cross-sectional views of yet another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIGS. 12A and 12B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIGS. 13A and 13B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIGS. 14A-14E are cross-sectional views of yet another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIGS. 15A and 15B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIGS. 16A and 16B are cross-sectional views of another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
- FIG. 17A is a perspective view of an exemplary embodiment of a thermal cycling device with the cover in a closed position in accordance with the present teachings;
- FIG. 17B is a partial perspective view of the exemplary embodiment of the thermal cycling device of FIG. 17B with the cover in an open position;
- FIGS. 18A and 18B show temperature profiles corresponding to a thermal cycling device cover, reaction chamber, and cap in accordance with various exemplary embodiments of the present teachings.
- FIG. 19 shows sequencing data resulting from performing the Example set forth herein.
- automatically actuating refers to causing a delivery device to expel a reagent that is being held by the delivery device to a reaction site without an operator having to open a vessel or other device in which a reaction is taking place and introduce a reagent (e.g., via pipetting, syringe, and/or various other invasive delivery techniques) to the reaction site.
- automatically actuating the delivery device may include causing the delivery device to introduce reagent to the reaction site without exposing the reaction to the surrounding environment from which it is substantially sealed or isolated (for example, at a first level of isolation) during the reaction.
- the reaction can remain substantially sealed or isolated at the first level of isolation.
- automated actuating may include an operator manually taking an action, such as depressing a switch, pulling a lever, etc., to cause the delivery device to dispense (expel) a reagent held by the delivery device to a reaction site.
- the operator need not expose the reaction taking place within a closed device, such as, for example, in a microtiter plate within a thermal cycler device, to the environment external to the closed device or otherwise remove a first level of isolation of the reaction.
- sample means any biological substance that contains cells and/or matter contained in cells. Samples also may contain such cellular matter mixed with other substances, such as, for example, buffers, reagents, and other substances that may react with the cellular matter or may be added to support a future reaction with the cellular matter.
- PCR / sequencing refers to a method for preparing a sample for determining a nucleotide sequence of DNA, the method including PCR amplifying the DNA, followed by one or more sequencing reactions.
- PCR/sequencing can include sequencing reactions repeated (or cycled) several times.
- Cycle sequencing is similar to PCR in that the sequencing reaction may use a thermostable DNA polymerase and the reaction is allowed to proceed, for example, at about 50°C to about 72°C, after which the double-stranded DNA is denatured at about 90 0 C to about 95°C, and then the oligonucleotide primer is annealed and extended at about 55 °C to about 72 °C.
- the cycle may be repeated from about 2 to about 25 times.
- phosphorothioate linkage refers to an inter- nucleotide linkage comprising a sulfur atom in place of a non-bridging oxygen atom within the phosphate linkages of a sugar phosphate backbone.
- the term phosphorothioate linkage refers to both phosphorothioate inter-nucleotide linkages and phosphorodithioate inter-nucleotide linkages.
- a "phosphorothioate linkage at a terminal 3' end” refers to a phosphorothioate linkage at the 3' terminus, that is, the last phosphate linkage of the sugar phosphate backbone at the 3' terminus.
- phosphodiester linkage refers to the linkage - PO 4 - which is used to link nucleotide monomers. Phosphodiester linkages as contemplated herein are linkages found in naturally-occurring DNA.
- the term "primer” refers to an oligonucleotide, typically between about 10 to 100 nucleotides in length, capable of selectively binding to a specified target nucleic acid or "template” by hybridizing with the template.
- the primer can provide a point of initiation for template-directed synthesis of a polynucleotide complementary to the template, which can take place in the presence of appropriate enzyme(s), cofactors, substrates such as nucleotides and oligonucleotides and the like.
- sequencing primer refers to an oligonucleotide primer that is used to initiate a sequencing reaction performed on a nucleic acid.
- sequencing primer refers to both a forward sequencing primer and to a reverse sequencing primer.
- amplification primer refers to an oligonucleotide capable of annealing to an RNA or DNA region adjacent a target sequence and serving as an initiation primer for DNA synthesis under suitable conditions well known in the art.
- a PCR reaction employs a pair of amplification primers including an "upstream” or “forward” primer and a “downstream” or “reverse” primer, which delimit a region of the RNA or DNA to be amplified.
- amplifying refers to a process whereby a portion of a nucleic acid is replicated. Unless specifically stated, “amplifying” refers to a single replication or to an arithmetic, logarithmic, or exponential amplification.
- determining a nucleotide base sequence or the term “determining information about a sequence” encompasses “sequence determination” and also encompasses other levels of information such as eliminating one or more possibilities for a sequence. It is noted that performing sequence determination of a polynucleotide typically yields equivalent information regarding the sequence of a perfectly complementary (100% complementary) polynucleotide and thus is equivalent to sequence determination performed directly on a perfectly complementary polynucleotide.
- nucleic acid sequence can refer to the nucleic acid material itself and is not restricted to the sequence information (i.e. the succession of letters chosen among the five base letters A, C, G, T, or U) that biochemically characterizes a specific nucleic acid, for example, a DNA or RNA molecule. Nucleic acids shown herein are presented in a 5' ⁇ 3' orientation unless otherwise indicated.
- polynucleotide refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages.
- a polynucleotide such as an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5' ⁇ 3' order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted.
- the letters A, C, G, and T can be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.
- the inter-nucleoside linkage is typically a phosphodiester bond, and the subunits are referred to as "nucleotides.”
- Oligonucleotide primers comprising other inter-nucleoside linkages, such as phosphorothioate linkages, are used in certain embodiments of the teachings. It will be appreciated that one or more of the subunits that make up such an oligonucleotide primer with a non- phosphodiester linkage can not comprise a phosphate group.
- nucleotide As used herein, and nucleic acids comprising one or more inter-nucleoside linkages that are not phosphodiester linkages are still referred to as "polynucleotides”, “oligonucleotides”, etc.
- sequence determination includes determination of partial as well as full sequence information. That is, the term includes sequence comparisons, fingerprinting, and like levels of information about a target polynucleotide, as well as the express identification and ordering of each nucleoside of the target polynucleotide within a region of interest.
- sequence determination comprises identifying a single nucleotide, while in other embodiments more than one nucleotide is identified. Identification of nucleosides, nucleotides, and/or bases are considered equivalent herein. It is noted that performing sequence determination on a polynucleotide typically yields equivalent information regarding the sequence of a perfectly complementary polynucleotide and thus is equivalent to sequence determination performed directly on a perfectly complementary polynucleotide.
- references to templates, oligonucleotides, primers, etc. generally mean populations or pools of nucleic acid molecules that are substantially identical within a relevant region rather than single molecules.
- a "template” generally means a plurality of substantially identical template molecules
- a “primer” generally means a plurality of substantially identical primer molecules, and the like.
- Cycle sequencing involves adding to a target nucleic acid or an amplification product thereof, sequencing primer, deoxynucleotide triphosphates (dNTPs), dye-labeled chain terminating nucleotides (e.g.,dideoxynucleotide triphosphates (ddNTPs-dyes)), and DNA polymerase, followed by thermal cycle sequencing.
- dNTPs deoxynucleotide triphosphates
- dye-labeled chain terminating nucleotides e.g.,dideoxynucleotide triphosphates (ddNTPs-dyes)
- DNA polymerase e.g.,ddNTPs-dyes
- reagent when reference is made to a "reagent,” it should be understood that a reagent is not necessarily limited to a single active component. Rather, a “reagent” can refer to a composition comprising multiple active components or a single active component.
- reagent addition after an initial reaction stage such as, for example, PCR
- reagent addition step may occur automatically and without the need for the operator to perform the reagent addition step in an invasive manner, for example, by exposing the reaction to the external environment or otherwise removing a first level of isolation of the reaction. This may minimize the risk of contamination, reduce labor needed to perform reaction, and/or increase overall throughput and efficiency.
- Increasing throughput and efficiency may be beneficial, for example, in various DNA detection applications, such as, for example, sequencing, mutation detection, copy number determination, genotyping, methylation detection, and/or other diagnostic or screening assays.
- Various exemplary embodiments of the present teachings may thus permit operators to begin a multi-stage reaction and not have to be present to enable the reaction to proceed to a next stage by performing one or more reagent addition steps that may be required.
- Various exemplary embodiments in accordance with the present teachings allow for automated (“hands-off") reagent addition after an initial stage of a multi-stage reaction, such as, for example, after PCR has been completed.
- devices and methods of the present teachings may be suitable for performing PCR/cycle sequencing without the need to open the reaction chamber after PCR thermal cycling to add the reagents (e.g., SAP (shrimp alkaline phosphatase) and exonuclease) used in the cleanup step and to open the reaction chamber again after the cleanup step to add sequencing reagents (e.g., primers and dNTPs).
- reagents e.g., SAP (shrimp alkaline phosphatase) and exonuclease
- sequencing reagents e.g., primers and dNTPs
- exemplary embodiments may be suitable for performing PCR/cycle sequencing, other multi-stage reactions may be performed as well, including, for example, other multi-stage reactions involving an initial PCR stage (e.g., a PCR/OLA reaction).
- exemplary embodiments in accordance with the present teachings may be used to deliver reagent after an initial PCR stage.
- exemplary embodiments may permit a single, automated reagent addition step after PCR, with the single reagent addition step supporting both the cleanup stage and the sequencing stage.
- the methods and devices of the present teachings may be used to deliver a reagent composition in a single, automated step after PCR, wherein the reagent comprises a nuclease and a nuclease-resistant sequencing primer.
- the reagent delivered in a single, automated step after PCR may comprise dye-labeled dideoxynucleotides triphosphates, exonuclease I, and an exonuclease-resistant sequencing primer.
- FIG. 2 illustrates a PCR/sequencing workflow in which a single reagent addition step (Step 2 in FIG. 2) follows the PCR stage (Step 1 in FIG. 2).
- the workflow of FIG. 2 and exemplary reagent compositions suitable for use with various exemplary reagent delivery methods and devices of the present teachings to perform PCR/sequencing reactions are described in US Patent Application 12/365,140, which is incorporated by reference.
- the reagent mixture delivered may include two universal primers resistant to exonuclease I, one of which may be equipped with a dragchute to extend the sequencing ladder past the end of the ladder from the first primer.
- Automated reagent delivery after an initial thermal cycling stage of a multistage reaction may pose challenges in that some reagents are temperature-sensitive and can become inactivated when subject to elevated temperatures.
- delivery mechanisms that hold reagent and are positioned proximate to where a reaction with elevated temperatures is occurring may also experience elevated temperatures that could render the reagent inactive prior to the desired addition of the reagent to the reaction.
- Various exemplary embodiments in accordance with the present teachings therefore, may minimize the risk of rendering such temperature- sensitive reagents inactive.
- heat-stable reagents may be used in the reagent delivery devices, which may not pose a concern of becoming inactive when heated to elevated temperatures.
- Various exemplary embodiments in accordance with the present teachings also may provide reagent delivery devices and methods configured for use with thermal cycling devices that typically have a block configured to support a sample holder (such as, for example, a microtiter plate) containing sample to be processed. The block is configured to be heated and cooled to cycle the sample in the sample holder through various temperatures for various time periods.
- thermal cycling devices typically also include a cover that is placed over the sample holder, which in conventional applications may be heated to prevent condensation of the reaction mixture in the sample holder.
- An exemplary embodiment of one such thermal cycling device that may be used, or modified for use, in conjunction with the present teachings includes the VeritiTM Thermal Cycler sold by Applied Biosystems. Those having skill in the art are familiar with various conventional thermal cycling devices and thus further details regarding such devices are not described herein.
- reagent delivery devices configured to hold reagents in a pattern that corresponds to the pattern of reaction sites, for example, reaction chambers (e.g., wells) of a microtiter plate, capillary tubes, reaction sites on a microcard, and/or other arrayed configurations of conventional sample holders used in various life sciences applications, such as PCR, and with which those having ordinary skill in the art are familiar.
- such arrayed formats may include an array of 24, 48, 96, or 384 reaction sites (e.g., wells of a microtiter plate) of a sample holder with which those ordinarily skilled in the art are familiar and delivery devices in accordance with various exemplary embodiments may hold reagents in a substantially corresponding arrayed format.
- the reagent delivery device 300 may include a support 305 that holds a plurality of beads 310 containing one or more reagents desired to be delivered to a reaction.
- the beads 310 may be a material that is solid or semi-solid below a threshold temperature, such as, for example, a gel or a wax.
- the reagent for which delivery to a reaction is desired may be contained within the beads 310.
- the beads 310 may be made of a material that is configured to melt upon reaching a predetermined temperature, for example, a temperature associated with a stage of a multi-stage reaction at which it is desired to introduce the one or more reagents contained in the beads 310.
- a predetermined temperature for example, a temperature associated with a stage of a multi-stage reaction at which it is desired to introduce the one or more reagents contained in the beads 310.
- Suitable solid and semi-solid materials that may be used for the beads include, but are not limited to, agarose gel (e.g., a low-melt agarose gel such as, for example, Sigma agarose type VII), a water-insoluble wax (e.g., that has a melting temperature of about 40 0 C for various exemplary embodiments), and/or an aqueous gel having a relatively defined gelling and melting transition temperature (i.e., exhibiting little or no hysteresis between those two temperatures).
- a suitable gel may also may melt at about 40 0 C for various exemplary embodiments described herein.
- Selecting a material for the bead that exhibits little or no hysteresis may facilitate the reagent delivery process when using a reagent that is temperature-sensitive since the temperature for solidifying (e.g., gelling) the material may be significantly lower than the temperature for melting it.
- the gelling temperature may be about 30 0 C while the melting temperature is about 65 0 C.
- Using a substance for the beads that has a relatively high melting temperature may pose problems if the temperature required to melt the bead is sufficiently high that the reagent contained in the bead become heat-denatured during the melting process.
- a concentrated solution of agarose e.g., a low-melt agarose gel
- a concentrated solution of agarose may be mixed and heated until it dissolves, for example, at about 95 0 C. This solution may then be cooled to about 35 0 C before it gels.
- the agarose solution may then be added to the reagent solution, aliquotted into beads and cooled to form "beads", allowing the heat-labile reagent to remain active.
- Another approach for forming the beads 310 in accordance with various exemplary embodiments may include injecting the reagent to be delivered into a low-melt wax in its solidified, beaded configuration.
- the beads 310 may be arrayed on the support 305 in a pattern that corresponds to the pattern of reaction sites, e.g., wells of a microtiter plate, capillary tubes, reaction sites on a microcard, and/or other arrayed configurations of conventional sample holders used in various life sciences applications, such as PCR, and with which those having ordinary skill in the art are familiar.
- arrayed formats may include an array of 24, 48, 96, or 384 reaction sites with which those ordinarily skilled in the art are familiar.
- the delivery device 300 may be positioned relative to such a sample holder such that the beads 310 align respectively with the reaction chambers so as to drop from the device 300 into the reaction mixture.
- the beads 310 may have a volume ranging from about 2 microliters to about 50 microliters.
- the beads 310 may have a volume of about 2 microliters to about 50 microliters, for example, about 6 microliters.
- the support 305 may comprise a plastic film, such as, for example, a film configured to create a seal over a conventional microtiter plate to prevent cross-contamination between wells and evaporation of substance within the wells.
- a plastic film such as, for example, a film configured to create a seal over a conventional microtiter plate to prevent cross-contamination between wells and evaporation of substance within the wells.
- a suitable plastic film that may be used for support 305 includes MicroAmpTM Clear Adhesive Film (part number 430631 1 ) sold by Applied Biosystems.
- the support 305 may be disposable such that after use for delivering the reagent in beads 310 to the desired reaction, the support 305 may be thrown away.
- the support 305 is substantially planar, supports having slightly indented regions to hold the beads 310 may also be used.
- FIGS. 4A-4C an exemplary embodiment of using the delivery device 300 for performing a PCR/sequencing reaction is schematically depicted.
- FIGS. 4A-4C show a partial, cross-sectional view of the delivery device 300 in cooperation with a microtiter plate 400 seated in a sample block 430 of a thermal cycling device.
- FIGS. 4A-4C depict only a single reaction chamber 405 in the microtiter plate 400 and the corresponding portions of the delivery device 300 and thermal cycling device that are in alignment with that reaction chamber 405 during a PCR/sequencing reaction workflow.
- the delivery device 300 may include an array of beads 310 arranged on a support 305.
- the beads 310 may be about 6 microliters each and may be formed of gel or wax containing a reagent composition comprising, for example, a dye- labeled dideoxynucleotide triphosphate, exonuclease I, and an exonuclease-resistant sequencing primer, such a reagent composition being described in more detail in US Patent Application 12/365,140, which is incorporated by reference.
- the beads 310 may comprise a gel (e.g., an agarose gel) or wax that melts at a temperature in a range of about 40 0 C to about 65 0 C.
- the melting temperature may be toward the upper end of that range without risking rendering the reagent inactive.
- the lower end of the range may be more suitable, though not required, for delivering temperature-sensitive reagents, such as are typically used, for example, for PCR/sequencing assays.
- the delivery device 300 may be positioned relative to a sample holder 400 such that each of the beads 310 aligns substantially with a reaction chamber 405 (e.g., well) of the sample holder 400.
- a standard PCR mixture 410 which may include, for example, thermostable DNA polymerase, dNTPs, magnesium ion, and buffer, and with which those ordinarily skilled in the art are familiar, may be placed in the reaction chambers 405 of the microtiter plate 400.
- a layer 415 of a substance may be placed on top of the PCR mixture 410, the purpose of which is to, among other things, prevent evaporation of the PCR mixture 410 during thermal cycling.
- the PCR mixture 410 may have a volume ranging from about 1 microliter to about 10 microliters.
- the volume of the PCR mixture 410 contained in each reaction chamber 405 may range from about 2 microliters to about 10 microliters, for example from about 5 microliters to about 10 microliters.
- a smaller PCR volume may enhance sequencing, as a smaller PCR volume facilitates overwhelming PCR nucleotides with cycle sequencing nucleotides.
- pipetting (or other dispensing) of smaller volumes may also need to be considered when choosing an initial PCR volume.
- the PCR mixture may contain about 1 microliter of master mix, 0.5 microliters of primers, and about 0.5 microliters of nucleic acid (e.g., DNA).
- the layer 415 may have a volume ranging from about 5 microliters to about 10 microliters.
- the specific gravity of the substance forming layer 415 may be less than water such that the layer 415 remains on top of the PCR mixture 410.
- the layer 415 may comprise any suitable substance, such as, for example, oil, that can be used to prevent evaporation of the PCR mixture 410 during thermal cycling.
- suitable substances such as, for example, oil
- Other desirable characteristics of suitable substances that can be used for layer 415 may include: insolubility in water, non- dissolving of detergents, relatively low viscosity to facilitate pipetting and/or other dispensing, relatively high boiling point (e.g., above about 250 0 C), inertness to PCR, nontoxic, and relatively inexpensive.
- suitable substances for layer 415 include, but are not limited to, for example, silicone oil, hydrocarbons (e.g., hexadecane), and/or mineral oil.
- the sample holder 400 having its reaction chambers 405 filled with the PCR mixture 410 and layer 415, along with the delivery device 300 placed relative thereto may be placed on a conventional sample block 430 of a thermal cycling device which is used to heat and cool the reaction chambers 405.
- a cover 435 of the thermal cycling device may be used to close the thermal cycling device and, when in the closed position, may be in contact with the delivery device 300, as shown in FIGS. 4A-4C.
- the cover 435 in alternative embodiments may be spaced from the delivery device 300 as long as sufficient heat may be transferred thereto to accomplish release of the beads 310 from the delivery device 300.
- the thermal cycling device may then be operated in accordance with a standard PCR protocol to heat the PCR mixture 410 in the reaction chambers 405.
- the cover 435 of the thermal cycling device may remain unheated or may be cooled so as to maintain the beads 310 at a temperature in which they remain intact and adhered to the support 305.
- the cover 435 may be maintained at a temperature ranging from about 20 0 C to about 40 0 C, for example, about 25 0 C, in order to prevent the beads 435 from melting. Keeping the cover 435 unheated or cooled differs from some conventional PCR techniques in which the cover of the thermal cycling device is heated to prevent condensation of the PCR mixture. In the exemplary embodiment of FIGS.
- the layer 415 may serve to seal the PCR reaction in the chambers 405 rather than relying on having the cover 435 heated.
- An additional benefit to keeping the cover 435 at a relatively lower temperature during the PCR reaction may be to hinder rendering inactive any temperature-sensitive reagents that are contained in the beads 310.
- the cover 435 may be heated to bring it up to at least the melting temperature of the beads 310, for example, to about 50 0 C to about 70 0 C in the case of agarose gel beads 310. Heating the cover 435 in turn causes the beads 310 to melt, thereby releasing the beads 310 and the reagents therein from the support 305 and into the PCR reaction product mixture 41 1 , as shown in FIG. 4B.
- the thermal cycling device may be used to thermal cycle the mixture in the reaction chambers 405 to achieve cleanup and sequencing, as desired.
- incubation of the exonuclease I at about 37 0 C for about 30 minutes may occur followed by heat-killing the enzyme at about 80 0 C for about 15 minutes.
- a standard cycle sequencing protocol may then be performed, with the cover 435 being heated to a temperature ranging from about 25 0 C to about 105 0 C, as depicted in FIG. 4C, and may be followed by, for example, ethanol precipitation, rehydration, and electrophoretic separation and detection to determine the sequence.
- Further details regarding the protocols to achieve sequencing once the reagent addition step of FIG. 4B has occurred may be found with reference US Patent Application 12/365,140, which is incorporated by reference. [076]
- 4A-4C illustrate an exemplary automated technique for delivery of reagent during a reaction (e.g., PCR/sequencing) that does not require an operator to be present to add desired reagents after the reaction has begun.
- a reaction e.g., PCR/sequencing
- Those having ordinary skill in the art would appreciate various modifications to the procedure described with reference to FIGS. 4A-4C to perform other reactions, such as, for example, other multi-stage reactions requiring reagent delivery after an initial reaction stage.
- One such modification may include providing more than one bead 310 having differing melting temperatures at a location on the support 305 corresponding to a respective reaction chamber 405.
- each bead 310 at a location may contain differing reagents and heating the cover 435 to differing temperatures may permit the beads 310 at each location to melt at different times, thus enabling sequential reagent addition to occur during the multi-stage reaction.
- the size of the beads 310 and the size of the reaction chambers 405 may be limiting factors in the number of beads 310 that may be positioned per location on the support 305 corresponding to a respective reaction chamber 405.
- FIGS. 5-7 illustrate other exemplary embodiments for reagent delivery in accordance with the present teachings. As will be explained in more detail below, the exemplary embodiments of FIGS. 5-7 may be used in lieu of the delivery device 300 described above with reference to FIGS. 3 and 4.
- FIG. 5A depicts a perspective view of a dispenser element 500 that may be used for automated reagent delivery in accordance with exemplary aspects of the present teachings.
- FIG. 5B depicts a perspective cross-sectional view of the dispenser element 500 of FIG. 5A taken through line 5B-5B in FIG. 5A.
- the dispenser element 500 may be hollow and have a cross-section that decreases from a first end 501 (e.g., the top end in the orientation of FIGS. 5A and 5B) to a second opposite end 502 (e.g., the bottom end in the orientation of FIGS. 5A and 5B).
- the hollow portion of the dispenser element 500 may form a chamber 505.
- the chamber 505 may have a length that extends through the length of the dispenser element 500 such that the chamber 505 defines openings 503 and 504 at the first end 501 and second end 502, respectively, of the dispenser element 500.
- the volume of the chamber 505 may range from about 5 microliters to about 50 microliters, for example, the volume of the chamber 505 may be about 18 microliters.
- the length of the chamber 505 from the first end 501 to the second end 502 of the dispenser element 500 may range from about 5 millimeters to about 10 millimeters, for example about 7 millimeters.
- the chamber 505 may have a cross-section that is larger in the region of the dispenser element 500 that extends from the first end 501 to about mid-way of the dispenser element 500 than in the region that extends from the second end 502 to about mid-way of the dispenser element 500.
- the cross-section of the chamber 505 may be tapered at about mid-way of the dispenser element 500 to transition between the larger cross-section and the smaller cross-section.
- the chambers 505 may have a substantially circular cross-section with a diameter of about 4 millimeters at the larger region tapering to about 0.5 millimeters at the smaller region.
- the chamber 505 may have various other cross-sectional configurations, such as, for example, oval, square, rectangular, triangular, hexagonal, pentagonal, etc. As will be explained further below, the differing cross-sections of the chamber 505 may permit substance retained in the chamber 505 to be held within the chamber 505 and prevented from exiting via gravity through the smaller cross-sectional portion of the chamber 505 and out opening 504 due to capillary forces acting on the substance.
- the portion of the dispenser element 500 having the larger outer cross-section in FIGS. 5A and 5B may be configured to be inserted into a friction-fit relationship with cap 601 of a strip 600 of caps 601 , as illustrated in the exemplary embodiment of FIG. 6.
- a cap strip that may be used is a MicroAmpTM 8-Cap Strip sold by Applied Biosystems.
- a typical format for such a cap strip 600 shown in FIG. 6 includes eight caps 601 so as to be compatible with the format of standard microtiter plates.
- Those ordinarily skilled in the art would recognize that other formats for a cap strip could be used, including any number of such caps ranging from one to more than one, without departing from the scope of the present teachings.
- the caps 601 may have a substantially dome-shaped closed end 602 (e.g., upper end in the orientation shown in FIG. 6) and the end 603 opposite the closed end 602 may be open so as to receive the dispenser element 500.
- the dome shape of the closed end 602 may confer various desirable features, as will be set forth below, other configurations also may be suitable and within the scope of the present teachings.
- the closed end 602 may be substantially flat.
- the dispenser element 500 may be made of a plastic material, such as, for example, Turcite®, Turcite XTM, or polypropylene, and the cap 601 may be made of polypropylene.
- both the cap 601 and the dispenser element 500 may be molded from polypropylene.
- FIG. 7 depicts, in cross-section, an exemplary embodiment of a dispenser element 500 inserted into a cap 601 , the chamber 505 of the dispenser element 500 being filled with a reagent 510 for which delivery to a reaction chamber 705 (which may for example be a well of a microtiter plate) is desired.
- the reagent 510 may be introduced into the chamber 505 so as to be held substantially within the tapered region 508 of the dispenser element. Capillary forces within the smaller cross-sectional region 507 of the dispenser element 500 may act to retain the reagent 510 within the chamber 505.
- a layer 515 of a volatile substance such as, for example, hexamethyldisiloxane having a boiling point of about 100 0 C, may be placed above the reagent 510 within the chamber 505 so as to fill the portion of the chamber 505 above where the reagent 510 resides, e.g., substantially within the larger cross-sectional portion 506 of the chamber 505.
- Other suitable substances for layer 515 may be substances that are substantially immiscible with the reagent 510, insoluble in water, and that don't evaporate at room temperature.
- the region above the surface of the volatile layer 515 and up to the inner surface of the dome-shaped end 602 of the cap 600 may contain air, thereby creating an air gap 520.
- the volume of reagent 510 may be about 6 microliters
- the volume of the volatile layer 515 may be about 15 microliters of silicone oil with a viscosity of about 0.65 centiStokes
- the volume of the air above the volatile layer 515 may be about 35 microliters.
- the cap 601 of FIG. 7 may be heated to a temperature sufficient to expand the air in the air gap and/or to volatize the volatile substance layer 515. Such expansion and/or volatization may increase the pressure within the chamber 505 above the reagent 510 causing the reagent 510 to be expelled from the dispenser element 500 and out of the opening 504.
- the cap 601 may be heated to a temperature ranging from about 80 0 C to about 105 0 C.
- the air in the air gap 520 may insulate the reagent 510 the heat directed to the cap 601 during the expelling step.
- FIG. 8 depicts an exemplary embodiment of using the assembly of FIG. 7 to deliver the reagent 510 to a reaction chamber 805.
- a single dispenser element 500, cap 601 , and reaction chamber 805 is illustrated in FIG. 8, it should be understood that there may be a plurality of such reaction chambers 805, such as, for example, wells of a multi-well microtiter plate of standard format with which those of skill in the art are familiar.
- a plurality of cap strips holding a plurality of corresponding dispenser elements may be respectively inserted into a plurality of rows of a sample holder.
- FIG. 7 The assembly of FIG. 7 is therefore inserted into the reaction chamber 805 such that the cap 601 is received in and seals the open end of the reaction chamber 805.
- the dispenser element 500 and cap 601 may be used to add reagent to the reaction chamber 805 during a multi-stage reaction.
- the reagent 510 may be a composition comprising, for example, a dye-labeled dideoxynucleotide triphosphates, exonuclease I, an exonuclease-resistant sequencing primer, and other Sanger sequencing reagent components (enzymes, dNTPs, etc.), or another reagent composition disclosed in the exemplary embodiments found in US Patent Application 12/365,140, which is incorporated by reference.
- the reaction chamber 805 may be filled with a PCR reaction mixture 810 and a layer 815 of a nonvolatile substance may rest on top the PCR reaction mixture 810.
- the layer 815 may be a substance like that described above with reference to layer 415 in the exemplary embodiment of FIGS. 4A-4C. Moreover, the volumes for the PCR reaction mixture 810 and the layer 815 may be similar to those described above with reference to the exemplary embodiment of FIGS. 4A-4C.
- the cap 601 may be heated to a temperature sufficient to cause the reagent 510 to be expelled from the dispenser element 500, as described above, and in turn to deliver the reagent 510 to the reaction chamber 805 and the PCR reaction products therein.
- the contents of the reaction chamber 805 can be subjected to an appropriate thermal cycling protocol to ultimately achieve cycle sequencing of the amplified PCR reaction products in the reaction chamber 805.
- the sequencing reaction may be followed with various post-process steps, such as, for example, ethanol precipitation, re-hydration, and sequence detection, as desired.
- the cover of a thermal cycling device used to perform the multi-stage reaction may remain unheated or cooled.
- a dispenser element having a structure similar to dispenser element 500 and/or a cap having a structure similar to cap 601 to actuate the delivery (e.g., expel) reagent therefrom and to achieve automated (“hands-off") delivery of the reagent at an appropriate time during a reaction, including, for example a multi-stage reaction such as a PCR/sequencing reaction.
- a multi-stage reaction such as a PCR/sequencing reaction.
- FIGS. 9-16 depict a single dispenser element inserted into a single reaction chamber, as with various exemplary embodiments described above, it should be understood that there may be a plurality of dispenser element/cap assemblies, for example, in conjunction with one or more cap strips, and a sample holder comprising an array of reaction chambers into which the plurality of dispenser element/cap assemblies are inserted to deliver reagent thereto.
- the overall structure of the dispenser elements, caps, reaction chambers and thermal cycling device cover are similar to those described above with reference to FIGS. 5-8, the reference numerals of those elements are maintained in FIGS. 9-16 for simplicity.
- various parts of the thermal cycling device are not shown, but those having ordinary skill in the art would understand how to use the exemplary embodiments of FIGS. 9-16 with thermal cycling devices having substantially conventional configurations.
- the dispenser element 500 is shown inserted into a cap 601 with a substantially dome-shaped closed end 602.
- the cap 601 including the closed end 602, may be made of a material that deforms upon heating to above a threshold temperature.
- a cover 435 on the thermal cycler device may be heated, causing the dome-shaped closed end 602 of the cap 601 to deform (e.g., collapse) under the weight of the cover 435, as depicted in FIG. 9B.
- the cover 435 may be spring-biased toward the dispenser element, as depicted schematically via the spring 905 in FIGS. 1 OA and 10B.
- the closed end 602 of the cap 601 may resist the force of the cover 435 and spring 905 acting to deform the cap 601.
- the force of the spring 905 and cover 435 may cause the closed end 602 of the cap 601 to collapse.
- the entire cap 601 , or at least the closed end 602 may be made of a material that either softens or shrinks upon heating to about a threshold temperature.
- the cap 601 , or at least the closed end 602 may be made of a plastic material, such as, for example, a polymer, that is substantially rigid up to about 50 0 C and that softens and becomes deformable so as to collapse upon reaching a sufficient temperature (e.g., "softening point").
- a plastic material such as, for example, a polymer, that is substantially rigid up to about 50 0 C and that softens and becomes deformable so as to collapse upon reaching a sufficient temperature (e.g., "softening point").
- Some heat-softening materials that may be suitable include, but are not limited to, various polymers used to protect electronics from overheating, as disclosed, for example, in U.S. Patent No. 6,896,824 and International Patent Publication WO 20040038732, both of which are incorporated by reference herein
- the entire cap 601 , or at least the upper closed end portion 602 thereof may be made of a heat shrink material.
- the cap 601 may be heated to at or above a threshold temperature, causing the closed end portion 602 to collapse (via shrinking).
- Suitable heat shrink materials that may be used to form the cap 601 and/or the closed end portion 602 of the cap 601 , include, but are not limited to, for example, polyolefin, fluoropolymer, PVC, neoprene, silicone, and Viton.
- suitable heat shrink materials may exhibit one or more of the following: biocompatibility, shrinking below about 100 0 C, and being substantially clear.
- Cesar-Scott, Inc. sells a polyolefin tubing (CSHS-10130) that is clear and shrinks at about 90 0 C and may be suitable for use to form the cap 601 and/or the upper portion 602 thereof.
- CSHS-10130 polyolefin tubing
- FIGS. 1 1 A and 11 B an exemplary embodiment of using a mechanical plunger mechanism to collapse the closed end 602 of the cap 601 is depicted.
- the plunger mechanism 1 105 may be associated with the cover 435 of a thermal cycling device and may move downward relative thereto at an appropriate time to collapse the upper end 602 of the cap 601. Collapsing the closed end 602 of the cap 601 may increase the pressure in the chamber 505 above the reagent 510 causing the reagent 510 to be expelled from the opening 504 of the dispenser element 500 and into the reaction chamber 805.
- a plurality of plunger mechanisms 1 105 may be associated with the cover 435 at positions that substantially align with positions of a plurality of dispenser elements 500 inserted in a strip of caps 601.
- the plurality of pistons 1 105 may be configured to be independently or simultaneously actuated to move downward to respectively depress the closed ends 602 of the caps 601.
- the plunger mechanism 1 105 may be actuated manually via an actuation mechanism disposed external to the thermal cycling device.
- the plunger mechanism 1 105 may be actuated automatically through a control system, for example, either at a predetermined time and/or upon reaching a predetermined temperature during a reaction.
- a control system for example, either at a predetermined time and/or upon reaching a predetermined temperature during a reaction.
- FIGS. 12A and 12B illustrate another exemplary embodiment that relies on a plunger mechanism 1205 to collapse the closed end 602 of the cap 601 to expel the reagent 510 from the dispenser element 500.
- a band 1210 comprising a heat shrink material (e.g., including any of the heat shrink materials described above with reference to the embodiment of FIGS. 10A and 10B) may be wrapped around the plunger mechanism 1205 and secured to the cap 601 , for example, via adhesive or other suitable securing mechanism, as shown in FIG. 12A.
- the cover 435 of a thermal cycling device may come into contact with or be disposed proximate to the band 1210 during a reaction.
- cover 435 may be heated, which may in turn heat the band 1210. Heating the band 1210 to or above a threshold temperature may cause the heat shrink material from which the band 1210 is made to shrink around the plunger mechanism 1205. The shrinking of the band 1210 may exert a force on the plunger mechanism 1205 in a direction toward the reaction chamber 805, causing the closed end 602 of the cap 601 to deform and collapse. The collapsing of the closed end 602 in turn may increase the pressure in the chamber 505 thereby expelling the reagent 510 out of the opening 504 of the dispenser element 500 and into the reaction chamber 805.
- FIGS. 13A and 13B depict a mechanism that may be used in conjunction with various exemplary embodiments described herein that rely on a collapsible closed end 602 of the cap 601.
- a plug member 1305 may be configured to be inserted into the opening 504 of the dispenser element 500 to seal the opening 504 to minimize the risk of the reagent 510 leaking out of the opening 504 and/or to prevent contamination of the chamber 505 prior to when it is desired to deliver the reagent 510.
- the plug member 1305 may be connected to an end of a rod 1315.
- the end of the rod 1315 opposite to the end at which the plug member 1305 is connected may be connected to the inner surface of the closed end 602 of the cap 601.
- the rod 1315 pushes the plug member 1305 out of the opening 504 to permit the reagent 510 to be expelled from the dispenser element 500 and into the reaction chamber 805.
- FIGS. 14A-14E depict an exemplary embodiment of using a plunger mechanism to release a series of individual reagent amounts 510a-510d (although four individual reagent amounts are depicted in FIGS. 14A-14E, any number of individual reagent amounts may be used and considered within the scope of the present teachings).
- the individual reagent amounts may be the same or may differ from each other (e.g. at least one individual reagent amount may differ in some way, such as composition, amount, etc. from another individual reagent amount).
- the exemplary embodiment of FIGS. 14A-14E may be useful for delivering differing reagents at differing times during a multi-stage reaction without the need for an operator to oversee the delivery of such reagents.
- the exemplary embodiment of FIGS. 14A-14E may include a dispenser element 500 wherein the dispenser element is not inserted into a cap. Instead, the opening 503 of the dispenser element 500 may be uncovered and configured to receive a plunger mechanism 1405. Individual reagent amounts 51 Oa-51 Od may be formed and held within the chamber 505 of the dispenser element 500 underneath the plunger mechanism 1405 such that the plunger mechanism 1405 contacts the individual reagent amount located on the top of the plurality of individual reagent amounts. In the exemplary embodiment of FIGS. 14A-14E, the plunger mechanism 1405 is in contact with individual reagent amount 51 Od, as shown in FIGS. 14A-14D. In the exemplary embodiment of FIGS.
- the individual reagent amounts 51 Oa-51 Od may be in the form of lyophilized pellets.
- FIGS. 14A-14E show pellets having a substantially rectangular cross-section, those ordinarily skilled in the art would recognize that the pellets could have a variety of configurations without departing from the scope of the present teachings.
- a separating medium may ensure that consecutive reagent amounts 51 Oa-51 Od are not accidentally delivered simultaneously during the same reagent addition step and/or to assist in maintaining the individual reagent amounts dry prior to delivery.
- the plunger mechanism 1405 may be associated with or situated below a cover 435 of the thermal cycling device, similar to that described above with reference to FIGS. 10A-10B.
- the plunger mechanism 1405 may be configured to be controlled in a stepwise manner to incrementally move the plunger mechanism 1405 in a downward direction away from the cover 435.
- Each incremental actuation and downward movement of the plunger mechanism 1405 may cause the individual reagent amount 510a, 510b, 51 Oc, or 51 Od that is positioned closest to the opening 504 of the dispenser element 500 to be expelled from the dispenser element 500 and into the reaction chamber 805.
- the plunger mechanism 1405 may be controlled to incrementally move in a downward direction expelling in a sequential manner and one at a time each individual reagent amount 51 Oa (FIG. 14B), 51 Ob (FIG. 14C), 51 Oc (FIG. 14D) and 51 Od (FIG. 14E) from the dispenser element 500 and into the reaction chamber 805.
- the plunger mechanism 1405 may be manually or automatically actuated to expel the individual reagent amounts 510a-510d as desired during a reaction.
- a reagent delivery device like in the exemplary embodiment of FIGS. 14A- 14E may permit a multi-stage reaction that requires more than one reagent addition step to be achieved in an automated manner without the need to open the reaction chamber and add reagents at each desired stage of the reaction for which a different reagent may be required.
- a conventional PCR/sequencing reaction requiring two reagent addition steps after the initial PCR reaction stage e.g., the addition of exonuclease in one stage and the addition of SAP in another
- FIGS. 15A and 15B Another exemplary embodiment that includes a dispenser element 500 and cap 601 with a closed end 602 is depicted in FIGS. 15A and 15B.
- the hollow portion of the cap 601 , including the closed end 602 may be filled with a material configured to expand, for example, upon being heated to or above a threshold temperature.
- the expandable material 1505 prior to heating, is disposed within the hollow interior of the closed end 602 of the cap 601.
- the cover 435 of the thermal cycling device may be in contact or close proximity to the closed end 602 and when it is desired to introduce the reagent 510 from the dispenser element 500 into the reaction chamber 805, the cover 435 may be heated so as to increase the temperature of the expandable material 1505 to at least the threshold temperature.
- the expandable material 1505 Upon heating to or above the threshold temperature, the expandable material 1505 expands, as shown in FIG. 15B, and enters the chamber 505 of the dispenser element 500 through the opening 503.
- the increased pressure in the chamber 505 due to the material 1505 entering the chamber 505 upon expansion causes the reagent 510 to be expelled from the dispenser element 500 and into the reaction chamber 850.
- Suitable materials for use as expandable material 1505 may include, for example, various foams, materials made of nanoparticles, and/or expandable microspheres.
- expandable material 1505 may be used as expandable material 1505.
- the expandable material 1505 may expand upon being heated to a temperature in a range from about 70 0 C to about 150 0 C.
- the cap 601 When used for reagent addition during a PCR/sequencing reaction, or for any multi-stage reaction in which reagent addition is desired after an initial stage having an elevated temperature requirement, it may be necessary, as described herein with respect to various other exemplary embodiments, to maintain the cap 601 at a temperature lower than the threshold temperature at which the expandable material 1505 expands. In various exemplary embodiments in accordance with the present teachings, this may be accomplished by having the cover 435 of the thermal cycling device unheated or cooled.
- FIGS. 16A and 16B Another exemplary embodiment for reagent delivery is depicted in FIGS. 16A and 16B.
- the reagent 510 may be contained in a containment structure 1620, which may, for example, have an inverted cup configuration.
- the containment structure 1620 with the reagent 510 therein may be adhered via a meltable material to a support 1605, such as, for example, a plastic film as described above with reference to the exemplary embodiment of FIGS. 4A-4C.
- the meltable material may reach or exceed a threshold melting temperature, thereby causing the meltable material to melt and release the cup 1620 and the reagent 510 therein into a reaction chamber 805 with which the cup 1620 is in cooperation.
- the cup 1620 may be similar in structure to a cap of a strip of caps like the cap strip 600 depicted in FIG. 6.
- Providing the reagent 510 within a containment structure that is adhered to the support 1605, rather than directly to the support 1605 may help provide sufficient weight to release the reagent 510 from the support 1605 during delivery. Without such a weighting mechanism, it may be more difficult to release the reagent from the film upon melting the meltable material. Since such a cup would itself be delivered into the reaction along with the reagent, a suitable material may be inert and nonreactive with the reaction components.
- the reagent 510 may be held within the containment structure 1620 via a material that may be meltable, such as, for example, a meltable wax, gel or other material, upon reaching a sufficient threshold temperature to release the reagent 510 from the containment structure 1620 during delivery.
- the containment structure 1620 may be sized and arranged to hold the reagent 510 within the structure via capillarity until the reagent 510 and containment structure 1620 are delivered.
- the reagent 510 is held within the containment structure 1620 prior to delivery, upon delivery to the reaction site, the reagent 510 may be exposed to the products therein and thereby be release into the reaction.
- a plurality of containment structures 1620 may be adhered to the support 1605 in a format such that the containment structures 1620 are placed in conjunction with one or more rows of reaction chambers 805 in an arrayed sample holder, such as, for example, a microtiter plate.
- an air gap may be provided above the reagent 510 to the inner surface of the upper end portion 602 of the cap 601. The air gap may insulate the reagent 510 from heat during the expelling of the reagent 510 from the dispenser element 500 so as to minimize the risk of deactivating the reagent 510 if the reagent 510 is temperature-sensitive.
- a layer of oil may be provided in the dispenser element 500 above the reagent 510 to coat the inner surface so the dispenser and facilitate delivery of the reagent.
- a layer of oil may be useful to facilitate delivery of relatively small volumes of liquid reagent by preventing capillary forces from causing the reagent to "stick" to the inner surfaces of the dispenser element 500.
- the oil may not be needed for the exemplary embodiment of FIGS. 14A-14E, as the reagent used in that embodiment may be in lyophilized form.
- An exemplary, non-limiting oil that may be used is silicone oil having a viscosity of about 5 centiStokes and in a volume ranging from about 20 microliters to about 50 microliters.
- silicone oil having a viscosity ranging from about 0.5 centiStokes to about 5 centi Stokes and a boiling point ranging from about 100 0 C to over about 250 0 C may be used.
- FIG. 17A shows an exemplary embodiment of a thermal cycling device 1700 modified with a fan 1750 mounted over the cover 1735. Ventilation holes 1760 may be made through the cover 1735 so as to allow the fan 1750 to exhaust heat from underneath the cover 1735 out of the thermal cycling device 1700.
- the cover 1735 may be desirable to cool the cover 1735 to a temperature ranging from about 25 0 C to about 40 0 C to maintain the temperature of the reagent inside the caps at a temperature of less than about 50 0 C, thereby minimizing the risk of rendering a temperature-sensitive reagent inactive and/or causing the reagent to be delivered too early in delivery devices that utilize heat as the mechanism by which to deliver the reagent.
- the fan 1750 may be turned off and the cover 1735 may be heated to reach a temperature sufficient to carry out remaining portions of a multi-stage reaction relying on thermal cycling and/or to release the reagent from the delivery device.
- the cover 1735 may be heated to a temperature ranging from about 50 0 C to about 105 0 C, for example about 105 0 C, after PCR has been completed.
- FIG. 17B depicts the thermal cycling device 1700 with the cover 1735 in an open position revealing a sample block 1730 which may be configured to receive, for example, a microtiter plate.
- Various exemplary embodiments of the present teachings also may include a thermal cycling device that is configured to be programmed so as to control the temperature of both the sample block and the cover.
- a thermal cycling device that is configured to be programmed so as to control the temperature of both the sample block and the cover.
- the sample block is controlled so as to cycle through a predetermined time-temperature program and the temperature of the cover is typically either heated to a single predetermined set temperature or left unheated.
- a controller may be used to program the thermal cycling device to perform PCR with the sample block cycling through various temperatures associated with a PCR cycle and with which those having ordinary skill in the art are familiar and the cover either left unheated or optionally, with the fan on, if desired), then to heat the cover (and. if previously on, turn the fan off) to a temperature sufficient to accomplish reagent delivery in accordance with various exemplary embodiments of the present teachings, and then control the temperature of the sample block in conjunction with optionally heating the cover or leaving the cover unheated to perform cycle sequencing.
- FIGS. 18A and 18B show temperature profiles of the cover, the well of a microAmpTM Optical 96-well reaction plate (Applied Biosystems part number 4306737), and the inside of the cap (Applied Biosystems part number N801 1535), wherein a thermal cycle was performed with the caps of a cap strip inserted into the wells of a microtiter plate.
- the wells were filled with 4 ul of PCR reaction mixture (primers (0.2 micromolars each), PCR master mix (1 x), and human genomic DNA (2 nanograms)) which was overlaid with 10 microliters of 5 centiStoke silicone oil.
- the temperature profiles in FIG. 18A correspond to a thermal cycle performed using a standard VeritiTM thermal cycler sold by Applied Biosystems wherein the cover remains unheated during the thermal cycle.
- the thermal cycle corresponding to the results shown in FIG. 18A was carried out using the following thermal cycle: 95 0 C for 10 minutes; 35 cycles of 96 0 C for 3 seconds, 62 0 C for 3 seconds, and 68 0 C for 8 seconds.
- the temperature inside the cap rises to 50 0 C.
- the temperature profiles in FIG. 18B correspond to the same thermal cycle as for FIG.
- the temperature inside the cap remains below 4O 0 C during the thermal cycle. Maintaining the temperature below 40 0 C may be useful when delivering a temperature-sensitive reagent, such as, for example, exonuclease I which degrades at about 50 0 C.
- a temperature-sensitive reagent such as, for example, exonuclease I which degrades at about 50 0 C.
- Maintaining the temperature of the cap at a relatively cool temperature may also be useful when using a cap made of a heat- shrinking and/or heat-softening material and/or when using a delivery mechanism that melts at a threshold temperature (e.g., an agarose or wax bead), in order to prevent premature reagent delivery.
- a threshold temperature e.g., an agarose or wax bead
- PCR and sequencing reactions were performed in a microAmpTM Optical 96-well reaction plate (Applied Biosystems part number 4306737) using a VertiTM 96-WeII Thermal Cycler, 200 microliter format (Applied Biosystems part number 4375786).
- Six wells of the 96-well plate contained 4 ul of PCR reaction mixture (primers (0.2 micromolars each), PCR master mix (1 x), and human genomic DNA (2 nanograms)) which was overlaid with 10 microliters of 5 centiStoke silicone oil.
- the PCR master mix used was AmpliTaq Gold® Fast PCR Mastermix, UP (Applied Biosystems part number 4390941 ).
- Exonuclease I digestion, exonuclease I heat-kill, and cycle sequencing was then carried out using the following thermal cycle: 37 0 C for 15 minutes; 80 0 C for 15 minutes; 96 0 C for 1 minute; 25 cycles of 96 0 C for 10 seconds, 50 0 C for 5 seconds, and 60 0 C for 75 seconds; and 4 0 C for an indefinite time until the next processing step is desired.
- reagent delivery devices set forth herein may be configured to be disposable or may be configured for multiple uses with appropriate cleaning between uses.
- the reagent delivery devices may be provided to users in an empty form such that users can customize the reagent they want to add to the device for delivery.
- the reagent delivery devices in various exemplary embodiments may be prefilled with preselected reagent and/or volatile layer, if applicable, and provided to users with preselected reagents therein that may be used for predetermined reaction protocols.
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Abstract
A method for delivering reagent during a thermal cycling reaction may include placing a delivery device containing a reagent proximate to a reaction site, initiating a first stage of the reaction, wherein the first stage includes a period of an elevated temperature, and after completing the first stage of the reaction, dispensing the reagent from the delivery device to the reaction site, wherein the dispensing includes automatically actuating the delivery device to deliver the reagent.
Description
DEVICES AND METHODS FOR REAGENT DELIVERY
TECHNICAL FIELD
[001] The present teachings relate to devices and methods for delivering reagents during a reaction. In particular, the present teachings relate to devices and methods for automated reagent delivery during a reaction.
INTRODUCTION
[002] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.
[003] Various biochemical and/or chemical reaction workflows (e.g., assays) require numerous stages in which controlling parameters of the reaction differ. For example, differing stages of a reaction may include differing temperatures, times, addition and/or removal of supporting reagents and other substances from the reaction, and/or other parameters that may need to be altered during the reaction. Often, conventional workflows involved in performing a multi-stage reaction involve various manual steps.
[004] For example, a conventional polymerase chain reaction (PCR)/sequencing workflow generally includes three stages, each of which requires reagent addition. Those stages include an initial PCR stage, a cleanup stage, and a sequencing stage. The PCR stage involves amplification of a template polynucleotide using amplification primers and a thermo-stable DNA polymerase enzyme. The cleanup stage is commonly performed by adding exonuclease I and alkaline phosphatase, followed by incubation, and subsequent heat-kill to inactivate the enzymes. After the cleanup stage, sequencing primers and reagents, such as, for example, dNTPs (deoxynucleotide
triphospates), ddNTPs-dyes (dideoxynucleotide triphospates-dyes), and polymerase may be added and a thermal cycle employed to perform sequencing. A standard PCR/sequencing workflow is illustrated in FIG. 1.
[005] Typical PCR uses an excess of amplification primers, some of which remain even upon completion of the reaction. Further, the PCR reaction products may contain excess dNTPs. Because excess amplification primers and excess dNTPs can interfere with a subsequent sequencing reaction, they are generally removed in a cleanup stage after the PCR reaction through the addition of exonuclease I and alkaline phosphatase enzymes. After an appropriate incubation period in which the exonuclease I degrades single-stranded DNA in the mixture after PCR and the alkaline phosphatase enzymes dephosphorylates dNTPs, the exonuclease I and alkaline phosphatase enzymes are subject to a heat-kill step to inactivate those enzymes. After that inactivation, sequencing reagents, including, for example, sequencing primers, dNTPs and ddNTPs-dyes are added to the remaining mixture and sequencing is performed.
[006] Thus, the conventional PCR/sequencing workflow described above involves two reagent addition steps after the PCR stage: (1 ) the reagent addition to remove the excess dNTPs and amplification primers and, thereafter, (2) the reagent addition to support sequencing. In conventional workflows, those reagent addition steps are done manually, typically by opening the vessel (or vessels) in which the reactions are taking place and adding the reagents.
[007] Another example of a multi-stage reaction that involves a reagent addition stage after performing PCR is PCR/oligonucleotide ligation assay (OLA). In a
conventional PCR/OLA workflow, a multiplex PCR reaction (with multiple primer pairs) is performed via thermal cycling, which may include a relatively long heat-kill step to kill the DNA polymerase (for example, at a temperature of about 95 0C for at least about 30 minutes.) After the heat-kill step, the reaction vessel is opened and thermostable ligase, ATP (adenosine-5'-triphosphate), dye-labeled and/or dragchute labeled probes are added (e.g., via pipetting or other manual, invasive mechanism). The reaction vessel is then closed and several cycles of cyclic ligation are performed (e.g., involving annealing, ligating, and denaturing). Once the several cycles are completed, the reaction vessel is opened and loaded onto a capillary electrophoresis instrument. Contrary to a PCR/sequencing workflow, the PCR/OLA workflow does not require a cleanup step after PCR. Moreover, unlike some reagents that may be used in PCR/sequencing workflows, in an exemplary process, the ligase used in PCR/OLA may be thermostable and therefore the risk of denaturing it (e.g., rendering it inactive) with heat before delivery is minimized.
[008] Various other biochemical and/or chemical multi-stage reaction workflows also may require the addition of reagent to the reaction mixture after an initial reaction stage, such as, for example, an initial PCR stage. The particular multi-stage reactions described generally above and in more detail below are exemplary and non-limiting. Those having ordinary skill in the art will recognize other assays that may require the addition of reagents at various stages of a reaction.
[009] Problems arise with conventional workflows for such multi-stage reactions, in particular, when one or more reagent addition steps are involved after an initial reaction stage (e.g., an initial PCR stage in the examples described above). Such
subsequent reagent addition steps, in particular when performed manually, may require extra labor, may increase the chance of introducing errors and/or contamination into the process, and may increase the overall time for performing the entire desired reaction. Moreover, multi-stage reactions wherein one or more stages requires a relatively elevated temperature (e.g., thermal cycling) may pose problems for reagents that are not heat-stable and can degrade upon reaching elevated temperatures. This is because keeping such reagents cool may be difficult if they are situated in the proximity of where the reaction is occurring.
SUMMARY
[010] The present invention may solve one or more of the above-mentioned problems. Other features and/or advantages may become apparent from the description which follows.
[01 1] According to various embodiments, the present teachings contemplate a method for delivering reagent during a thermal cycling reaction that includes placing a delivery device containing a reagent proximate to a reaction site, initiating a first stage of the reaction, wherein the first stage includes a period of an elevated temperature, and after completing the first stage of the reaction, dispensing the reagent from the delivery device to the reaction site, wherein the dispensing includes automatically actuating the delivery device to deliver the reagent.
[012] According to various embodiments, the present teachings contemplate a kit for sequencing nucleic acids that includes a reagent comprising a nuclease and a nuclease-resistant sequencing primer and a dispensing device holding the reagent,
wherein the dispensing device is configured to automatically deliver the reagent to a reaction site.
According to various embodiments, the present teachings contemplate a method of sequencing nucleic acids that includes positioning a delivery device holding a reagent proximate a reaction site, amplifying nucleic acids at the reaction site in a first reaction to form amplified nucleic acids, wherein the first reaction comprises thermal cycling. After the amplifying step, the method may include automatically actuating the delivery device to deliver the reagent to the reaction site and causing the amplified nucleic acids to react in a sequencing reaction with the reagent.
[013] Additional objects and advantages may be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Those objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
[014] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary embodiments of the present teachings and together with the description, serve to explain certain principles. In the drawings:
[016] FIG. 1 is a schematic diagram representing a conventional PCR/ sequencing workflow;
[017] FIG. 2 is a schematic diagram representing a PCR/sequencing workflow comprising two stages and utilizing a nuclease-resistant sequencing primer according to various exemplary embodiments of the present teachings;
[018] FIG. 3 is a plan view of an exemplary embodiment of a reagent delivery device in accordance with the present teachings;
[019] FIGS. 4A-4C show exemplary steps for delivering a reagent during a workflow using the reagent delivery device of FIG. 3;
[020] FIG. 5A is an exemplary embodiment of a dispenser element of a reagent delivery device in accordance with the present teaching;
[021] FIG. 5B is a cross-sectional view of the dispenser element taken through line 5B-5B in FIG. 5A;
[022] FIG. 6 is a perspective view of an exemplary embodiment of a strip cap;
[023] FIG. 7 is a cross-sectional view of an exemplary embodiment of a reagent delivery device in accordance with the present teachings;
[024] FIG. 8 is a cross-sectional view of the reagent delivery device of FIG. 7 in cooperation with a reaction chamber to deliver reagent thereto;
[025] FIGS. 9A and 9B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[026] FIGS. 10A and 10B are cross-sectional views of another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[027] FIGS. 11 A and 1 1 B are cross-sectional views of yet another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[028] FIGS. 12A and 12B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[029] FIGS. 13A and 13B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[030] FIGS. 14A-14E are cross-sectional views of yet another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[031] FIGS. 15A and 15B are cross-sectional views of an exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[032] FIGS. 16A and 16B are cross-sectional views of another exemplary embodiment of a reagent delivery device in cooperation with a reaction chamber for delivering reagent to the reaction chamber;
[033] FIG. 17A is a perspective view of an exemplary embodiment of a thermal cycling device with the cover in a closed position in accordance with the present teachings;
[034] FIG. 17B is a partial perspective view of the exemplary embodiment of the thermal cycling device of FIG. 17B with the cover in an open position;
[035] FIGS. 18A and 18B show temperature profiles corresponding to a thermal cycling device cover, reaction chamber, and cap in accordance with various exemplary embodiments of the present teachings; and
[036] FIG. 19 shows sequencing data resulting from performing the Example set forth herein.
DETAILED DESCRIPTION
[037] Reference will now be made in detail to various exemplary embodiments, some of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.
[038] To facilitate understanding of the present teachings, the following definitions are provided. It is to be understood that, in general, terms not otherwise defined are to be given their ordinary meanings or meanings as generally accepted in the art.
[039] As used herein, the term "automatically actuating" and variants thereof refers to causing a delivery device to expel a reagent that is being held by the delivery device to a reaction site without an operator having to open a vessel or other device in which a reaction is taking place and introduce a reagent (e.g., via pipetting, syringe,
and/or various other invasive delivery techniques) to the reaction site. In other words, automatically actuating the delivery device may include causing the delivery device to introduce reagent to the reaction site without exposing the reaction to the surrounding environment from which it is substantially sealed or isolated (for example, at a first level of isolation) during the reaction. Thus, during delivery of the reagent, the reaction can remain substantially sealed or isolated at the first level of isolation. It should be understood that the term "automatically actuating" may include an operator manually taking an action, such as depressing a switch, pulling a lever, etc., to cause the delivery device to dispense (expel) a reagent held by the delivery device to a reaction site. However, in such circumstances, the operator need not expose the reaction taking place within a closed device, such as, for example, in a microtiter plate within a thermal cycler device, to the environment external to the closed device or otherwise remove a first level of isolation of the reaction.
[040] As used herein, "sample" means any biological substance that contains cells and/or matter contained in cells. Samples also may contain such cellular matter mixed with other substances, such as, for example, buffers, reagents, and other substances that may react with the cellular matter or may be added to support a future reaction with the cellular matter.
[041] As used herein, the term "PCR / sequencing" refers to a method for preparing a sample for determining a nucleotide sequence of DNA, the method including PCR amplifying the DNA, followed by one or more sequencing reactions. Thus, in some cases, PCR/sequencing can include sequencing reactions repeated (or cycled) several times. Cycle sequencing is similar to PCR in that the sequencing
reaction may use a thermostable DNA polymerase and the reaction is allowed to proceed, for example, at about 50°C to about 72°C, after which the double-stranded DNA is denatured at about 90 0C to about 95°C, and then the oligonucleotide primer is annealed and extended at about 55 °C to about 72 °C. The cycle may be repeated from about 2 to about 25 times.
[042] As used herein, the term "phosphorothioate linkage" refers to an inter- nucleotide linkage comprising a sulfur atom in place of a non-bridging oxygen atom within the phosphate linkages of a sugar phosphate backbone. The term phosphorothioate linkage refers to both phosphorothioate inter-nucleotide linkages and phosphorodithioate inter-nucleotide linkages. A "phosphorothioate linkage at a terminal 3' end" refers to a phosphorothioate linkage at the 3' terminus, that is, the last phosphate linkage of the sugar phosphate backbone at the 3' terminus.
[043] As used herein, the term "phosphodiester linkage" refers to the linkage - PO4 - which is used to link nucleotide monomers. Phosphodiester linkages as contemplated herein are linkages found in naturally-occurring DNA.
[044] As used herein, the term "primer" refers to an oligonucleotide, typically between about 10 to 100 nucleotides in length, capable of selectively binding to a specified target nucleic acid or "template" by hybridizing with the template. The primer can provide a point of initiation for template-directed synthesis of a polynucleotide complementary to the template, which can take place in the presence of appropriate enzyme(s), cofactors, substrates such as nucleotides and oligonucleotides and the like.
[045] As used herein, the term "sequencing primer" refers to an oligonucleotide primer that is used to initiate a sequencing reaction performed on a nucleic acid. The
term "sequencing primer" refers to both a forward sequencing primer and to a reverse sequencing primer.
[046] As used herein, the term "amplification primer" refers to an oligonucleotide capable of annealing to an RNA or DNA region adjacent a target sequence and serving as an initiation primer for DNA synthesis under suitable conditions well known in the art. Typically, a PCR reaction employs a pair of amplification primers including an "upstream" or "forward" primer and a "downstream" or "reverse" primer, which delimit a region of the RNA or DNA to be amplified.
[047] As used herein, the term "amplifying" refers to a process whereby a portion of a nucleic acid is replicated. Unless specifically stated, "amplifying" refers to a single replication or to an arithmetic, logarithmic, or exponential amplification.
[048] As used herein, the term "determining a nucleotide base sequence" or the term "determining information about a sequence" encompasses "sequence determination" and also encompasses other levels of information such as eliminating one or more possibilities for a sequence. It is noted that performing sequence determination of a polynucleotide typically yields equivalent information regarding the sequence of a perfectly complementary (100% complementary) polynucleotide and thus is equivalent to sequence determination performed directly on a perfectly complementary polynucleotide.
[049] The term "nucleic acid sequence" as used herein can refer to the nucleic acid material itself and is not restricted to the sequence information (i.e. the succession of letters chosen among the five base letters A, C, G, T, or U) that biochemically
characterizes a specific nucleic acid, for example, a DNA or RNA molecule. Nucleic acids shown herein are presented in a 5' → 3' orientation unless otherwise indicated.
[050] As used herein, the terms "polynucleotide", "nucleic acid", or "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Whenever a polynucleotide such as an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5' → 3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes deoxythymidine, unless otherwise noted. The letters A, C, G, and T can be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art. In naturally occurring polynucleotides, the inter-nucleoside linkage is typically a phosphodiester bond, and the subunits are referred to as "nucleotides." Oligonucleotide primers comprising other inter-nucleoside linkages, such as phosphorothioate linkages, are used in certain embodiments of the teachings. It will be appreciated that one or more of the subunits that make up such an oligonucleotide primer with a non- phosphodiester linkage can not comprise a phosphate group. Such analogs of nucleotides are considered to fall within the scope of the term "nucleotide" as used herein, and nucleic acids comprising one or more inter-nucleoside linkages that are not phosphodiester linkages are still referred to as "polynucleotides", "oligonucleotides", etc.
[051] As used herein "sequence determination", "determining a nucleotide base sequence", "sequencing", and like terms include determination of partial as well as full sequence information. That is, the term includes sequence comparisons, fingerprinting,
and like levels of information about a target polynucleotide, as well as the express identification and ordering of each nucleoside of the target polynucleotide within a region of interest. In certain embodiments, "sequence determination" comprises identifying a single nucleotide, while in other embodiments more than one nucleotide is identified. Identification of nucleosides, nucleotides, and/or bases are considered equivalent herein. It is noted that performing sequence determination on a polynucleotide typically yields equivalent information regarding the sequence of a perfectly complementary polynucleotide and thus is equivalent to sequence determination performed directly on a perfectly complementary polynucleotide.
[052] As will be appreciated by one of ordinary skill in the art, references to templates, oligonucleotides, primers, etc., generally mean populations or pools of nucleic acid molecules that are substantially identical within a relevant region rather than single molecules. For example, a "template" generally means a plurality of substantially identical template molecules; a "primer" generally means a plurality of substantially identical primer molecules, and the like.
[053] Cycle sequencing involves adding to a target nucleic acid or an amplification product thereof, sequencing primer, deoxynucleotide triphosphates (dNTPs), dye-labeled chain terminating nucleotides (e.g.,dideoxynucleotide triphosphates (ddNTPs-dyes)), and DNA polymerase, followed by thermal cycle sequencing. Standard cycle sequencing procedures are well established. Cycle sequencing procedures are described in more detail, for example, in U.S. Patent No. 5,741 ,676, and U.S. Patent No. 5,756,285, each herein incorporated by reference in its entirety.
[054] As used herein, when reference is made to a "reagent," it should be understood that a reagent is not necessarily limited to a single active component. Rather, a "reagent" can refer to a composition comprising multiple active components or a single active component.
[055] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
[056] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a range of "less than 10" includes any and all subranges between (and including) the minimum value of zero and the maximum value of 10, that is, any
and all subranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 5.
[057] It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. Thus, for example, reference to "a biological sample" includes two or more different biological samples. As used herein, the term "include" and its grammatical variants are intended to be non- limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
[058] Various methods and devices in accordance with the present teachings permit the addition of reagent without an invasive delivery technique during a biochemical and/or chemical reaction, such as, for example, a multi-stage reaction. For example, reagent addition after an initial reaction stage, such as, for example, PCR, may occur automatically and without the need for the operator to perform the reagent addition step in an invasive manner, for example, by exposing the reaction to the external environment or otherwise removing a first level of isolation of the reaction. This may minimize the risk of contamination, reduce labor needed to perform reaction, and/or increase overall throughput and efficiency. Increasing throughput and efficiency may be beneficial, for example, in various DNA detection applications, such as, for example, sequencing, mutation detection, copy number determination, genotyping, methylation detection, and/or other diagnostic or screening assays.
[059] Various exemplary embodiments of the present teachings may thus permit operators to begin a multi-stage reaction and not have to be present to enable the
reaction to proceed to a next stage by performing one or more reagent addition steps that may be required. Various exemplary embodiments in accordance with the present teachings allow for automated ("hands-off") reagent addition after an initial stage of a multi-stage reaction, such as, for example, after PCR has been completed.
[060] In accordance with various exemplary embodiments, devices and methods of the present teachings may be suitable for performing PCR/cycle sequencing without the need to open the reaction chamber after PCR thermal cycling to add the reagents (e.g., SAP (shrimp alkaline phosphatase) and exonuclease) used in the cleanup step and to open the reaction chamber again after the cleanup step to add sequencing reagents (e.g., primers and dNTPs). Although exemplary embodiments may be suitable for performing PCR/cycle sequencing, other multi-stage reactions may be performed as well, including, for example, other multi-stage reactions involving an initial PCR stage (e.g., a PCR/OLA reaction).
[061] When performing a PCR/sequencing reaction, various exemplary embodiments in accordance with the present teachings may be used to deliver reagent after an initial PCR stage. For example, exemplary embodiments may permit a single, automated reagent addition step after PCR, with the single reagent addition step supporting both the cleanup stage and the sequencing stage. In accordance with various exemplary embodiments, the methods and devices of the present teachings may be used to deliver a reagent composition in a single, automated step after PCR, wherein the reagent comprises a nuclease and a nuclease-resistant sequencing primer. More specifically, the reagent delivered in a single, automated step after PCR may comprise dye-labeled dideoxynucleotides triphosphates, exonuclease I, and an
exonuclease-resistant sequencing primer. FIG. 2 illustrates a PCR/sequencing workflow in which a single reagent addition step (Step 2 in FIG. 2) follows the PCR stage (Step 1 in FIG. 2). The workflow of FIG. 2 and exemplary reagent compositions suitable for use with various exemplary reagent delivery methods and devices of the present teachings to perform PCR/sequencing reactions are described in US Patent Application 12/365,140, which is incorporated by reference. To accomplish bidirectional sequencing, the reagent mixture delivered may include two universal primers resistant to exonuclease I, one of which may be equipped with a dragchute to extend the sequencing ladder past the end of the ladder from the first primer.
[062] Automated reagent delivery after an initial thermal cycling stage of a multistage reaction, such as, for example, after PCR, may pose challenges in that some reagents are temperature-sensitive and can become inactivated when subject to elevated temperatures. Thus, delivery mechanisms that hold reagent and are positioned proximate to where a reaction with elevated temperatures is occurring (e.g., during PCR or other initial thermal cycling reaction) may also experience elevated temperatures that could render the reagent inactive prior to the desired addition of the reagent to the reaction. Various exemplary embodiments in accordance with the present teachings, therefore, may minimize the risk of rendering such temperature- sensitive reagents inactive. In various exemplary embodiments, however, heat-stable reagents may be used in the reagent delivery devices, which may not pose a concern of becoming inactive when heated to elevated temperatures.
[063] Various exemplary embodiments in accordance with the present teachings also may provide reagent delivery devices and methods configured for use with thermal
cycling devices that typically have a block configured to support a sample holder (such as, for example, a microtiter plate) containing sample to be processed. The block is configured to be heated and cooled to cycle the sample in the sample holder through various temperatures for various time periods. Such thermal cycling devices typically also include a cover that is placed over the sample holder, which in conventional applications may be heated to prevent condensation of the reaction mixture in the sample holder. An exemplary embodiment of one such thermal cycling device that may be used, or modified for use, in conjunction with the present teachings includes the Veriti™ Thermal Cycler sold by Applied Biosystems. Those having skill in the art are familiar with various conventional thermal cycling devices and thus further details regarding such devices are not described herein.
[064] Various exemplary embodiments of the present teachings may provide reagent delivery devices configured to hold reagents in a pattern that corresponds to the pattern of reaction sites, for example, reaction chambers (e.g., wells) of a microtiter plate, capillary tubes, reaction sites on a microcard, and/or other arrayed configurations of conventional sample holders used in various life sciences applications, such as PCR, and with which those having ordinary skill in the art are familiar. By way of example, such arrayed formats may include an array of 24, 48, 96, or 384 reaction sites (e.g., wells of a microtiter plate) of a sample holder with which those ordinarily skilled in the art are familiar and delivery devices in accordance with various exemplary embodiments may hold reagents in a substantially corresponding arrayed format.
[065] With reference now to FIG. 3, an exemplary embodiment of a reagent delivery device in accordance with the present teachings is depicted in plan view. The
reagent delivery device 300 may include a support 305 that holds a plurality of beads 310 containing one or more reagents desired to be delivered to a reaction. In various exemplary embodiments, the beads 310 may be a material that is solid or semi-solid below a threshold temperature, such as, for example, a gel or a wax. The reagent for which delivery to a reaction is desired may be contained within the beads 310. The beads 310 may be made of a material that is configured to melt upon reaching a predetermined temperature, for example, a temperature associated with a stage of a multi-stage reaction at which it is desired to introduce the one or more reagents contained in the beads 310. Thus, when placed in cooperation with a reaction chamber in which a reaction is taking place, upon a temperature of the reaction reaching the melting temperature of the beads 310, the beads 310 may melt, dropping off the surface 305 and into the reaction site (e.g., chamber) in which it is desired to introduce the reagent contained in the beads 310.
[066] Examples of suitable solid and semi-solid materials that may be used for the beads include, but are not limited to, agarose gel (e.g., a low-melt agarose gel such as, for example, Sigma agarose type VII), a water-insoluble wax (e.g., that has a melting temperature of about 40 0C for various exemplary embodiments), and/or an aqueous gel having a relatively defined gelling and melting transition temperature (i.e., exhibiting little or no hysteresis between those two temperatures). A suitable gel may also may melt at about 40 0C for various exemplary embodiments described herein. Selecting a material for the bead that exhibits little or no hysteresis may facilitate the reagent delivery process when using a reagent that is temperature-sensitive since the temperature for solidifying (e.g., gelling) the material may be significantly lower than the
temperature for melting it. For example, for Sigma VII agarose the gelling temperature may be about 30 0C while the melting temperature is about 65 0C. Using a substance for the beads that has a relatively high melting temperature may pose problems if the temperature required to melt the bead is sufficiently high that the reagent contained in the bead become heat-denatured during the melting process.
[067] To form the beads 310 in accordance with an exemplary embodiment in which agarose gel is used to contain the reagents, a concentrated solution of agarose (e.g., a low-melt agarose gel) may be mixed and heated until it dissolves, for example, at about 95 0C. This solution may then be cooled to about 35 0C before it gels. The agarose solution may then be added to the reagent solution, aliquotted into beads and cooled to form "beads", allowing the heat-labile reagent to remain active. Another approach for forming the beads 310 in accordance with various exemplary embodiments may include injecting the reagent to be delivered into a low-melt wax in its solidified, beaded configuration.
[068] In an exemplary embodiment, the beads 310 may be arrayed on the support 305 in a pattern that corresponds to the pattern of reaction sites, e.g., wells of a microtiter plate, capillary tubes, reaction sites on a microcard, and/or other arrayed configurations of conventional sample holders used in various life sciences applications, such as PCR, and with which those having ordinary skill in the art are familiar. By way of example, such arrayed formats may include an array of 24, 48, 96, or 384 reaction sites with which those ordinarily skilled in the art are familiar. In this way, the delivery device 300 may be positioned relative to such a sample holder such that the beads 310 align respectively with the reaction chambers so as to drop from the device 300 into the
reaction mixture. In various exemplary embodiments, the beads 310 may have a volume ranging from about 2 microliters to about 50 microliters. By way of example, for performing a PCR/sequencing reaction, the beads 310 may have a volume of about 2 microliters to about 50 microliters, for example, about 6 microliters.
[069] In various exemplary embodiments, the support 305 may comprise a plastic film, such as, for example, a film configured to create a seal over a conventional microtiter plate to prevent cross-contamination between wells and evaporation of substance within the wells. One example of a suitable plastic film that may be used for support 305 includes MicroAmp™ Clear Adhesive Film (part number 430631 1 ) sold by Applied Biosystems. The support 305 may be disposable such that after use for delivering the reagent in beads 310 to the desired reaction, the support 305 may be thrown away. Although, in the exemplary embodiments depicted in FIG. 3, the support 305 is substantially planar, supports having slightly indented regions to hold the beads 310 may also be used.
[070] Referring now to FIGS. 4A-4C, an exemplary embodiment of using the delivery device 300 for performing a PCR/sequencing reaction is schematically depicted. FIGS. 4A-4C show a partial, cross-sectional view of the delivery device 300 in cooperation with a microtiter plate 400 seated in a sample block 430 of a thermal cycling device. For ease of illustration, FIGS. 4A-4C depict only a single reaction chamber 405 in the microtiter plate 400 and the corresponding portions of the delivery device 300 and thermal cycling device that are in alignment with that reaction chamber 405 during a PCR/sequencing reaction workflow.
[071] As described above, the delivery device 300 may include an array of beads 310 arranged on a support 305. In an exemplary embodiment for performing a PCR/sequencing reaction, the beads 310 may be about 6 microliters each and may be formed of gel or wax containing a reagent composition comprising, for example, a dye- labeled dideoxynucleotide triphosphate, exonuclease I, and an exonuclease-resistant sequencing primer, such a reagent composition being described in more detail in US Patent Application 12/365,140, which is incorporated by reference. By way of example, the beads 310 may comprise a gel (e.g., an agarose gel) or wax that melts at a temperature in a range of about 40 0C to about 65 0C. For the delivery of thermostable reagents, such as are typically used in PCR/OLA assays, the melting temperature may be toward the upper end of that range without risking rendering the reagent inactive. The lower end of the range may be more suitable, though not required, for delivering temperature-sensitive reagents, such as are typically used, for example, for PCR/sequencing assays. The delivery device 300 may be positioned relative to a sample holder 400 such that each of the beads 310 aligns substantially with a reaction chamber 405 (e.g., well) of the sample holder 400.
[072] A standard PCR mixture 410, which may include, for example, thermostable DNA polymerase, dNTPs, magnesium ion, and buffer, and with which those ordinarily skilled in the art are familiar, may be placed in the reaction chambers 405 of the microtiter plate 400. A layer 415 of a substance may be placed on top of the PCR mixture 410, the purpose of which is to, among other things, prevent evaporation of the PCR mixture 410 during thermal cycling. In accordance with an exemplary embodiment, the PCR mixture 410 may have a volume ranging from about 1 microliter
to about 10 microliters. For example, in the case of the PCR mixture 410 containing one or more limiting reagents, including primers and/or dNTPs, the volume of the PCR mixture 410 contained in each reaction chamber 405 may range from about 2 microliters to about 10 microliters, for example from about 5 microliters to about 10 microliters. When choosing a PCR volume, a smaller PCR volume may enhance sequencing, as a smaller PCR volume facilitates overwhelming PCR nucleotides with cycle sequencing nucleotides. However, pipetting (or other dispensing) of smaller volumes may also need to be considered when choosing an initial PCR volume. For a 2 microliter volume, the PCR mixture may contain about 1 microliter of master mix, 0.5 microliters of primers, and about 0.5 microliters of nucleic acid (e.g., DNA). In an exemplary embodiment, the layer 415 may have a volume ranging from about 5 microliters to about 10 microliters. The specific gravity of the substance forming layer 415 may be less than water such that the layer 415 remains on top of the PCR mixture 410.
[073] In various exemplary embodiments, the layer 415 may comprise any suitable substance, such as, for example, oil, that can be used to prevent evaporation of the PCR mixture 410 during thermal cycling. Other desirable characteristics of suitable substances that can be used for layer 415 may include: insolubility in water, non- dissolving of detergents, relatively low viscosity to facilitate pipetting and/or other dispensing, relatively high boiling point (e.g., above about 250 0C), inertness to PCR, nontoxic, and relatively inexpensive. Those having ordinary skill in the art would recognize that a suitable substance for use in the exemplary embodiment of FIGS. 4A- 4C may not exhibit one or more of the above-listed desirable characteristics, yet may
nonetheless be suitable for use, and skilled artisans would understand how to select such a suitable substance based on the present teachings. Examples of suitable substances for layer 415 include, but are not limited to, for example, silicone oil, hydrocarbons (e.g., hexadecane), and/or mineral oil.
[074] The sample holder 400, having its reaction chambers 405 filled with the PCR mixture 410 and layer 415, along with the delivery device 300 placed relative thereto may be placed on a conventional sample block 430 of a thermal cycling device which is used to heat and cool the reaction chambers 405. A cover 435 of the thermal cycling device may be used to close the thermal cycling device and, when in the closed position, may be in contact with the delivery device 300, as shown in FIGS. 4A-4C. Those having skill in the art will appreciate, however, that the cover 435 in alternative embodiments may be spaced from the delivery device 300 as long as sufficient heat may be transferred thereto to accomplish release of the beads 310 from the delivery device 300. The thermal cycling device may then be operated in accordance with a standard PCR protocol to heat the PCR mixture 410 in the reaction chambers 405. During the standard PCR protocol, however, the cover 435 of the thermal cycling device may remain unheated or may be cooled so as to maintain the beads 310 at a temperature in which they remain intact and adhered to the support 305. In an exemplary embodiment, the cover 435 may be maintained at a temperature ranging from about 20 0C to about 40 0C, for example, about 25 0C, in order to prevent the beads 435 from melting. Keeping the cover 435 unheated or cooled differs from some conventional PCR techniques in which the cover of the thermal cycling device is heated to prevent condensation of the PCR mixture. In the exemplary embodiment of FIGS.
4A-4C, the layer 415 may serve to seal the PCR reaction in the chambers 405 rather than relying on having the cover 435 heated. An additional benefit to keeping the cover 435 at a relatively lower temperature during the PCR reaction may be to hinder rendering inactive any temperature-sensitive reagents that are contained in the beads 310.
[075] After completing the PCR reaction, the cover 435 may be heated to bring it up to at least the melting temperature of the beads 310, for example, to about 50 0C to about 70 0C in the case of agarose gel beads 310. Heating the cover 435 in turn causes the beads 310 to melt, thereby releasing the beads 310 and the reagents therein from the support 305 and into the PCR reaction product mixture 41 1 , as shown in FIG. 4B. Once the reagents from the beads 310 have been introduced into the PCR reaction product mixture 41 1 , the thermal cycling device may be used to thermal cycle the mixture in the reaction chambers 405 to achieve cleanup and sequencing, as desired. For example, incubation of the exonuclease I at about 37 0C for about 30 minutes may occur followed by heat-killing the enzyme at about 80 0C for about 15 minutes. A standard cycle sequencing protocol may then be performed, with the cover 435 being heated to a temperature ranging from about 25 0C to about 105 0C, as depicted in FIG. 4C, and may be followed by, for example, ethanol precipitation, rehydration, and electrophoretic separation and detection to determine the sequence. Further details regarding the protocols to achieve sequencing once the reagent addition step of FIG. 4B has occurred may be found with reference US Patent Application 12/365,140, which is incorporated by reference.
[076] Thus, FIGS. 4A-4C illustrate an exemplary automated technique for delivery of reagent during a reaction (e.g., PCR/sequencing) that does not require an operator to be present to add desired reagents after the reaction has begun. Those having ordinary skill in the art would appreciate various modifications to the procedure described with reference to FIGS. 4A-4C to perform other reactions, such as, for example, other multi-stage reactions requiring reagent delivery after an initial reaction stage. One such modification may include providing more than one bead 310 having differing melting temperatures at a location on the support 305 corresponding to a respective reaction chamber 405. In such an exemplary embodiment, each bead 310 at a location may contain differing reagents and heating the cover 435 to differing temperatures may permit the beads 310 at each location to melt at different times, thus enabling sequential reagent addition to occur during the multi-stage reaction. The size of the beads 310 and the size of the reaction chambers 405 may be limiting factors in the number of beads 310 that may be positioned per location on the support 305 corresponding to a respective reaction chamber 405.
[077] FIGS. 5-7 illustrate other exemplary embodiments for reagent delivery in accordance with the present teachings. As will be explained in more detail below, the exemplary embodiments of FIGS. 5-7 may be used in lieu of the delivery device 300 described above with reference to FIGS. 3 and 4.
[078] FIG. 5A depicts a perspective view of a dispenser element 500 that may be used for automated reagent delivery in accordance with exemplary aspects of the present teachings. FIG. 5B depicts a perspective cross-sectional view of the dispenser element 500 of FIG. 5A taken through line 5B-5B in FIG. 5A. The dispenser element
500 may be hollow and have a cross-section that decreases from a first end 501 (e.g., the top end in the orientation of FIGS. 5A and 5B) to a second opposite end 502 (e.g., the bottom end in the orientation of FIGS. 5A and 5B). The hollow portion of the dispenser element 500 may form a chamber 505. The chamber 505 may have a length that extends through the length of the dispenser element 500 such that the chamber 505 defines openings 503 and 504 at the first end 501 and second end 502, respectively, of the dispenser element 500. In various exemplary embodiments, the volume of the chamber 505 may range from about 5 microliters to about 50 microliters, for example, the volume of the chamber 505 may be about 18 microliters. The length of the chamber 505 from the first end 501 to the second end 502 of the dispenser element 500 may range from about 5 millimeters to about 10 millimeters, for example about 7 millimeters.
[079] The chamber 505 may have a cross-section that is larger in the region of the dispenser element 500 that extends from the first end 501 to about mid-way of the dispenser element 500 than in the region that extends from the second end 502 to about mid-way of the dispenser element 500. The cross-section of the chamber 505 may be tapered at about mid-way of the dispenser element 500 to transition between the larger cross-section and the smaller cross-section. By way of non-limiting example only, the chambers 505 may have a substantially circular cross-section with a diameter of about 4 millimeters at the larger region tapering to about 0.5 millimeters at the smaller region. Those having ordinary skill in the art will appreciate that the chamber 505 may have various other cross-sectional configurations, such as, for example, oval, square, rectangular, triangular, hexagonal, pentagonal, etc. As will be explained further below,
the differing cross-sections of the chamber 505 may permit substance retained in the chamber 505 to be held within the chamber 505 and prevented from exiting via gravity through the smaller cross-sectional portion of the chamber 505 and out opening 504 due to capillary forces acting on the substance.
[080] In various exemplary embodiments, the portion of the dispenser element 500 having the larger outer cross-section in FIGS. 5A and 5B may be configured to be inserted into a friction-fit relationship with cap 601 of a strip 600 of caps 601 , as illustrated in the exemplary embodiment of FIG. 6. Those having skill in the art are familiar with such cap strips 600 and the use of the same to cover wells in microtiter plates during conventional thermal cycling procedures. One exemplary embodiment of a cap strip that may be used is a MicroAmp™ 8-Cap Strip sold by Applied Biosystems. A typical format for such a cap strip 600 shown in FIG. 6 includes eight caps 601 so as to be compatible with the format of standard microtiter plates. Those ordinarily skilled in the art would recognize that other formats for a cap strip could be used, including any number of such caps ranging from one to more than one, without departing from the scope of the present teachings.
[081] As shown in the exemplary embodiment of FIG. 6, the caps 601 may have a substantially dome-shaped closed end 602 (e.g., upper end in the orientation shown in FIG. 6) and the end 603 opposite the closed end 602 may be open so as to receive the dispenser element 500. Although the dome shape of the closed end 602 may confer various desirable features, as will be set forth below, other configurations also may be suitable and within the scope of the present teachings. For example, the closed end 602 may be substantially flat.
[082] In various exemplary embodiments, the dispenser element 500 may be made of a plastic material, such as, for example, Turcite®, Turcite X™, or polypropylene, and the cap 601 may be made of polypropylene. In various exemplary embodiments, both the cap 601 and the dispenser element 500 may be molded from polypropylene.
[083] FIG. 7 depicts, in cross-section, an exemplary embodiment of a dispenser element 500 inserted into a cap 601 , the chamber 505 of the dispenser element 500 being filled with a reagent 510 for which delivery to a reaction chamber 705 (which may for example be a well of a microtiter plate) is desired. The reagent 510 may be introduced into the chamber 505 so as to be held substantially within the tapered region 508 of the dispenser element. Capillary forces within the smaller cross-sectional region 507 of the dispenser element 500 may act to retain the reagent 510 within the chamber 505. A layer 515 of a volatile substance, such as, for example, hexamethyldisiloxane having a boiling point of about 100 0C, may be placed above the reagent 510 within the chamber 505 so as to fill the portion of the chamber 505 above where the reagent 510 resides, e.g., substantially within the larger cross-sectional portion 506 of the chamber 505. Other suitable substances for layer 515 may be substances that are substantially immiscible with the reagent 510, insoluble in water, and that don't evaporate at room temperature. The region above the surface of the volatile layer 515 and up to the inner surface of the dome-shaped end 602 of the cap 600 may contain air, thereby creating an air gap 520. In accordance with one exemplary embodiment, the volume of reagent 510 may be about 6 microliters, the volume of the volatile layer 515 may be about 15
microliters of silicone oil with a viscosity of about 0.65 centiStokes, and the volume of the air above the volatile layer 515 may be about 35 microliters.
[084] To deliver the reagent 510, the cap 601 of FIG. 7 may be heated to a temperature sufficient to expand the air in the air gap and/or to volatize the volatile substance layer 515. Such expansion and/or volatization may increase the pressure within the chamber 505 above the reagent 510 causing the reagent 510 to be expelled from the dispenser element 500 and out of the opening 504. By way of example, the cap 601 may be heated to a temperature ranging from about 80 0C to about 105 0C. In circumstances in which the reagent 510 is temperature-sensitive and there is a risk of rendering the reagent 510 inactive upon heating it above a threshold temperature, the air in the air gap 520 may insulate the reagent 510 the heat directed to the cap 601 during the expelling step. Moreover, to avoid heat-denaturing the reagent 510 during delivery, it may be desirable to choose a substance for layer 515 and size of the chamber 505 such that only a portion (e.g., distal portion) of the layer 515 is volatized during delivery of the reagent 510.
[085] FIG. 8 depicts an exemplary embodiment of using the assembly of FIG. 7 to deliver the reagent 510 to a reaction chamber 805. Although only a single dispenser element 500, cap 601 , and reaction chamber 805 is illustrated in FIG. 8, it should be understood that there may be a plurality of such reaction chambers 805, such as, for example, wells of a multi-well microtiter plate of standard format with which those of skill in the art are familiar. Similarly, there may be a plurality of dispenser elements 500 inserted in caps 601 of a cap strip 600 (shown in FIG. 6) and the entire cap strip 600 may be inserted into a corresponding row of reactions chambers 805 (e.g., wells) of a
microtiter plate. Moreover, in various exemplary embodiments, a plurality of cap strips holding a plurality of corresponding dispenser elements may be respectively inserted into a plurality of rows of a sample holder.
[086] The assembly of FIG. 7 is therefore inserted into the reaction chamber 805 such that the cap 601 is received in and seals the open end of the reaction chamber 805. In a manner similar to that described with reference to FIGS. 4A-4C, the dispenser element 500 and cap 601 may be used to add reagent to the reaction chamber 805 during a multi-stage reaction. For example, for a PCR/sequencing reaction, the reagent 510 may be a composition comprising, for example, a dye-labeled dideoxynucleotide triphosphates, exonuclease I, an exonuclease-resistant sequencing primer, and other Sanger sequencing reagent components (enzymes, dNTPs, etc.), or another reagent composition disclosed in the exemplary embodiments found in US Patent Application 12/365,140, which is incorporated by reference. The reaction chamber 805 may be filled with a PCR reaction mixture 810 and a layer 815 of a nonvolatile substance may rest on top the PCR reaction mixture 810. The layer 815 may be a substance like that described above with reference to layer 415 in the exemplary embodiment of FIGS. 4A-4C. Moreover, the volumes for the PCR reaction mixture 810 and the layer 815 may be similar to those described above with reference to the exemplary embodiment of FIGS. 4A-4C.
[087] After an initial round of thermal cycling during which PCR occurs in the reaction chamber 805, the cap 601 may be heated to a temperature sufficient to cause the reagent 510 to be expelled from the dispenser element 500, as described above, and in turn to deliver the reagent 510 to the reaction chamber 805 and the PCR reaction
products therein. Upon delivery of the reagent 510 to the reaction chamber 805, the contents of the reaction chamber 805 can be subjected to an appropriate thermal cycling protocol to ultimately achieve cycle sequencing of the amplified PCR reaction products in the reaction chamber 805. As was described with reference to the exemplary embodiment of FIGS. 4A-4C, the sequencing reaction may be followed with various post-process steps, such as, for example, ethanol precipitation, re-hydration, and sequence detection, as desired.
[088] To prevent the heat from an initial reaction stage requiring an elevated temperature from causing the layer 515 to volatize and/or the air in the air gap 520 to expand and thus expel the reagent 510 and/or to minimize the risk of the heat inactivating a temperature-sensitive reagent, as with the exemplary embodiment of FIGS. 4A-4C, the cover of a thermal cycling device used to perform the multi-stage reaction may remain unheated or cooled. In an exemplary embodiment when used to perform a PCR/sequencing reaction, for example, it may be desirable to maintain the temperature of the cover of the thermal cycling device such that the temperature of the cap 601 is below about 50 0C.
[089] Various other mechanisms may be used in conjunction with a dispenser element having a structure similar to dispenser element 500 and/or a cap having a structure similar to cap 601 to actuate the delivery (e.g., expel) reagent therefrom and to achieve automated ("hands-off") delivery of the reagent at an appropriate time during a reaction, including, for example a multi-stage reaction such as a PCR/sequencing reaction. Exemplary embodiments of some such configurations are depicted schematically in FIGS. 9-16. Although the exemplary embodiments of FIGS. 9-16
depict a single dispenser element inserted into a single reaction chamber, as with various exemplary embodiments described above, it should be understood that there may be a plurality of dispenser element/cap assemblies, for example, in conjunction with one or more cap strips, and a sample holder comprising an array of reaction chambers into which the plurality of dispenser element/cap assemblies are inserted to deliver reagent thereto. Because the overall structure of the dispenser elements, caps, reaction chambers and thermal cycling device cover are similar to those described above with reference to FIGS. 5-8, the reference numerals of those elements are maintained in FIGS. 9-16 for simplicity. Also for simplicity, various parts of the thermal cycling device are not shown, but those having ordinary skill in the art would understand how to use the exemplary embodiments of FIGS. 9-16 with thermal cycling devices having substantially conventional configurations.
[090] With reference to FIG. 9A, the dispenser element 500 is shown inserted into a cap 601 with a substantially dome-shaped closed end 602. The cap 601 , including the closed end 602, may be made of a material that deforms upon heating to above a threshold temperature. In this way, after an initial reaction stage, such as, for example, PCR, a cover 435 on the thermal cycler device may be heated, causing the dome-shaped closed end 602 of the cap 601 to deform (e.g., collapse) under the weight of the cover 435, as depicted in FIG. 9B. The collapsing of the closed end 602 of the cap 601 in turn may increase the pressure in the chamber 505 above the reagent 510 and thereby expel the reagent 510 out of the opening 504 in the dispenser element 500 and into a reaction chamber 805.
[091] In an alternative configuration, the cover 435 may be spring-biased toward the dispenser element, as depicted schematically via the spring 905 in FIGS. 1 OA and 10B. Prior to deforming upon heating, the closed end 602 of the cap 601 may resist the force of the cover 435 and spring 905 acting to deform the cap 601. Upon being heated by the cover 435, however, the force of the spring 905 and cover 435 may cause the closed end 602 of the cap 601 to collapse.
[092] In the exemplary embodiments of FIGS. 9A-9B and 10A-1 OB, the entire cap 601 , or at least the closed end 602 may be made of a material that either softens or shrinks upon heating to about a threshold temperature. In various exemplary embodiments, the cap 601 , or at least the closed end 602, may be made of a plastic material, such as, for example, a polymer, that is substantially rigid up to about 50 0C and that softens and becomes deformable so as to collapse upon reaching a sufficient temperature (e.g., "softening point"). Some heat-softening materials that may be suitable include, but are not limited to, various polymers used to protect electronics from overheating, as disclosed, for example, in U.S. Patent No. 6,896,824 and International Patent Publication WO 20040038732, both of which are incorporated by reference herein in their entireties.
[093] In various exemplary embodiments, the entire cap 601 , or at least the upper closed end portion 602 thereof may be made of a heat shrink material. Upon heating the cover 435 associated with the thermal cycling device, the cap 601 may be heated to at or above a threshold temperature, causing the closed end portion 602 to collapse (via shrinking). Suitable heat shrink materials that may be used to form the cap 601 and/or the closed end portion 602 of the cap 601 , include, but are not limited to,
for example, polyolefin, fluoropolymer, PVC, neoprene, silicone, and Viton. In various exemplary embodiments, suitable heat shrink materials may exhibit one or more of the following: biocompatibility, shrinking below about 100 0C, and being substantially clear. Cesar-Scott, Inc. sells a polyolefin tubing (CSHS-10130) that is clear and shrinks at about 90 0C and may be suitable for use to form the cap 601 and/or the upper portion 602 thereof. Those having ordinary skill in the art would understand how to select an appropriate heat shrink material in accordance with the present teachings, with the above specific materials and characteristics being exemplary and non-limiting only.
[094] Those having skill in the art will appreciate that the configuration of the reagent 510 after being released from the delivery device 300 and into the contents of the reaction chamber that is depicted in FIGS. 9B and 1 OB and in other exemplary embodiments described below is schematic for ease of illustration and that the reagent 510 may dissolve when delivered to the reaction.
[095] With reference to FIGS. 1 1 A and 11 B, an exemplary embodiment of using a mechanical plunger mechanism to collapse the closed end 602 of the cap 601 is depicted. The plunger mechanism 1 105 may be associated with the cover 435 of a thermal cycling device and may move downward relative thereto at an appropriate time to collapse the upper end 602 of the cap 601. Collapsing the closed end 602 of the cap 601 may increase the pressure in the chamber 505 above the reagent 510 causing the reagent 510 to be expelled from the opening 504 of the dispenser element 500 and into the reaction chamber 805. A plurality of plunger mechanisms 1 105 may be associated with the cover 435 at positions that substantially align with positions of a plurality of dispenser elements 500 inserted in a strip of caps 601. The plurality of pistons 1 105
may be configured to be independently or simultaneously actuated to move downward to respectively depress the closed ends 602 of the caps 601.
[096] In various exemplary embodiments, the plunger mechanism 1 105 may be actuated manually via an actuation mechanism disposed external to the thermal cycling device. Alternatively, the plunger mechanism 1 105 may be actuated automatically through a control system, for example, either at a predetermined time and/or upon reaching a predetermined temperature during a reaction. Those having ordinary skill in the art would understand how to provide a control system that could be used to actuate the plunger mechanism 1105 as desired during a reaction to accomplish release of the reagent 510 from the dispenser element 500.
[097] FIGS. 12A and 12B illustrate another exemplary embodiment that relies on a plunger mechanism 1205 to collapse the closed end 602 of the cap 601 to expel the reagent 510 from the dispenser element 500. In the exemplary embodiment of FIGS. 12A and 12B, a band 1210 comprising a heat shrink material (e.g., including any of the heat shrink materials described above with reference to the embodiment of FIGS. 10A and 10B) may be wrapped around the plunger mechanism 1205 and secured to the cap 601 , for example, via adhesive or other suitable securing mechanism, as shown in FIG. 12A. The cover 435 of a thermal cycling device may come into contact with or be disposed proximate to the band 1210 during a reaction. When it is desired to deliver the reagent 510 into a reaction chamber 805, cover 435 may be heated, which may in turn heat the band 1210. Heating the band 1210 to or above a threshold temperature may cause the heat shrink material from which the band 1210 is made to shrink around the plunger mechanism 1205. The shrinking of the band 1210 may exert a force on the
plunger mechanism 1205 in a direction toward the reaction chamber 805, causing the closed end 602 of the cap 601 to deform and collapse. The collapsing of the closed end 602 in turn may increase the pressure in the chamber 505 thereby expelling the reagent 510 out of the opening 504 of the dispenser element 500 and into the reaction chamber 805.
[098] The exemplary embodiment of FIGS. 13A and 13B depict a mechanism that may be used in conjunction with various exemplary embodiments described herein that rely on a collapsible closed end 602 of the cap 601. Referring to FIG. 13A, a plug member 1305 may be configured to be inserted into the opening 504 of the dispenser element 500 to seal the opening 504 to minimize the risk of the reagent 510 leaking out of the opening 504 and/or to prevent contamination of the chamber 505 prior to when it is desired to deliver the reagent 510. The plug member 1305 may be connected to an end of a rod 1315. The end of the rod 1315 opposite to the end at which the plug member 1305 is connected may be connected to the inner surface of the closed end 602 of the cap 601. When the closed end 602 of the cap 601 collapses, as shown in FIG. 13B, the rod 1315 pushes the plug member 1305 out of the opening 504 to permit the reagent 510 to be expelled from the dispenser element 500 and into the reaction chamber 805.
[099] FIGS. 14A-14E depict an exemplary embodiment of using a plunger mechanism to release a series of individual reagent amounts 510a-510d (although four individual reagent amounts are depicted in FIGS. 14A-14E, any number of individual reagent amounts may be used and considered within the scope of the present teachings). Moreover, the individual reagent amounts may be the same or may differ
from each other (e.g. at least one individual reagent amount may differ in some way, such as composition, amount, etc. from another individual reagent amount). The exemplary embodiment of FIGS. 14A-14E may be useful for delivering differing reagents at differing times during a multi-stage reaction without the need for an operator to oversee the delivery of such reagents.
[0100] The exemplary embodiment of FIGS. 14A-14E may include a dispenser element 500 wherein the dispenser element is not inserted into a cap. Instead, the opening 503 of the dispenser element 500 may be uncovered and configured to receive a plunger mechanism 1405. Individual reagent amounts 51 Oa-51 Od may be formed and held within the chamber 505 of the dispenser element 500 underneath the plunger mechanism 1405 such that the plunger mechanism 1405 contacts the individual reagent amount located on the top of the plurality of individual reagent amounts. In the exemplary embodiment of FIGS. 14A-14E, the plunger mechanism 1405 is in contact with individual reagent amount 51 Od, as shown in FIGS. 14A-14D. In the exemplary embodiment of FIGS. 14A-14E, the individual reagent amounts 51 Oa-51 Od may be in the form of lyophilized pellets. Although FIGS. 14A-14E show pellets having a substantially rectangular cross-section, those ordinarily skilled in the art would recognize that the pellets could have a variety of configurations without departing from the scope of the present teachings. Further, when delivering multiple reagent types, it may be useful to separate each of the individual reagent amounts 51 Oa-51 Od by a separating medium, such as, for example, a plastic pellet that also gets delivered. Such a separating medium may ensure that consecutive reagent amounts 51 Oa-51 Od are not
accidentally delivered simultaneously during the same reagent addition step and/or to assist in maintaining the individual reagent amounts dry prior to delivery.
[0101 ] The plunger mechanism 1405 may be associated with or situated below a cover 435 of the thermal cycling device, similar to that described above with reference to FIGS. 10A-10B. The plunger mechanism 1405 may be configured to be controlled in a stepwise manner to incrementally move the plunger mechanism 1405 in a downward direction away from the cover 435. Each incremental actuation and downward movement of the plunger mechanism 1405 may cause the individual reagent amount 510a, 510b, 51 Oc, or 51 Od that is positioned closest to the opening 504 of the dispenser element 500 to be expelled from the dispenser element 500 and into the reaction chamber 805. Thus, as shown in FIGS. 14B-14E, the plunger mechanism 1405 may be controlled to incrementally move in a downward direction expelling in a sequential manner and one at a time each individual reagent amount 51 Oa (FIG. 14B), 51 Ob (FIG. 14C), 51 Oc (FIG. 14D) and 51 Od (FIG. 14E) from the dispenser element 500 and into the reaction chamber 805. As described above with reference to the exemplary embodiment of FIGS. 1 1 A- 11 B, the plunger mechanism 1405 may be manually or automatically actuated to expel the individual reagent amounts 510a-510d as desired during a reaction.
[0102] A reagent delivery device like in the exemplary embodiment of FIGS. 14A- 14E may permit a multi-stage reaction that requires more than one reagent addition step to be achieved in an automated manner without the need to open the reaction chamber and add reagents at each desired stage of the reaction for which a different reagent may be required. Thus, for example, a conventional PCR/sequencing reaction
requiring two reagent addition steps after the initial PCR reaction stage (e.g., the addition of exonuclease in one stage and the addition of SAP in another), as described herein, may be accomplished in an automated (e.g., "hands off") manner by utilizing the exemplary embodiment of FIGS. 14A-14E to deliver the exonuclease first and then later the SAP.
[0103] Another exemplary embodiment that includes a dispenser element 500 and cap 601 with a closed end 602 is depicted in FIGS. 15A and 15B. In this embodiment, the hollow portion of the cap 601 , including the closed end 602 may be filled with a material configured to expand, for example, upon being heated to or above a threshold temperature. In FIG. 15A, the expandable material 1505, prior to heating, is disposed within the hollow interior of the closed end 602 of the cap 601. The cover 435 of the thermal cycling device may be in contact or close proximity to the closed end 602 and when it is desired to introduce the reagent 510 from the dispenser element 500 into the reaction chamber 805, the cover 435 may be heated so as to increase the temperature of the expandable material 1505 to at least the threshold temperature. Upon heating to or above the threshold temperature, the expandable material 1505 expands, as shown in FIG. 15B, and enters the chamber 505 of the dispenser element 500 through the opening 503. The increased pressure in the chamber 505 due to the material 1505 entering the chamber 505 upon expansion causes the reagent 510 to be expelled from the dispenser element 500 and into the reaction chamber 850.
[0104] Suitable materials for use as expandable material 1505 may include, for example, various foams, materials made of nanoparticles, and/or expandable microspheres. By way of example, Expancel® expandable microspheres manufactured
by Sundsvall of Sweden may be used as expandable material 1505. In various exemplary embodiments, the expandable material 1505 may expand upon being heated to a temperature in a range from about 70 0C to about 150 0C. When used for reagent addition during a PCR/sequencing reaction, or for any multi-stage reaction in which reagent addition is desired after an initial stage having an elevated temperature requirement, it may be necessary, as described herein with respect to various other exemplary embodiments, to maintain the cap 601 at a temperature lower than the threshold temperature at which the expandable material 1505 expands. In various exemplary embodiments in accordance with the present teachings, this may be accomplished by having the cover 435 of the thermal cycling device unheated or cooled.
[0105] Another exemplary embodiment for reagent delivery is depicted in FIGS. 16A and 16B. With reference to FIG. 16A, the reagent 510 may be contained in a containment structure 1620, which may, for example, have an inverted cup configuration. The containment structure 1620 with the reagent 510 therein may be adhered via a meltable material to a support 1605, such as, for example, a plastic film as described above with reference to the exemplary embodiment of FIGS. 4A-4C. Upon heating, the meltable material may reach or exceed a threshold melting temperature, thereby causing the meltable material to melt and release the cup 1620 and the reagent 510 therein into a reaction chamber 805 with which the cup 1620 is in cooperation.
[0106] In an exemplary embodiment, the cup 1620 may be similar in structure to a cap of a strip of caps like the cap strip 600 depicted in FIG. 6. However, those having
ordinary skill in the art would understand that a variety of configurations may be utilized without departing from the scope of the present teachings. Providing the reagent 510 within a containment structure that is adhered to the support 1605, rather than directly to the support 1605 (e.g., as in the exemplary embodiments of FIGS. 4A-4C), may help provide sufficient weight to release the reagent 510 from the support 1605 during delivery. Without such a weighting mechanism, it may be more difficult to release the reagent from the film upon melting the meltable material. Since such a cup would itself be delivered into the reaction along with the reagent, a suitable material may be inert and nonreactive with the reaction components.
[0107] In various exemplary embodiments, the reagent 510 may be held within the containment structure 1620 via a material that may be meltable, such as, for example, a meltable wax, gel or other material, upon reaching a sufficient threshold temperature to release the reagent 510 from the containment structure 1620 during delivery. Alternatively, the containment structure 1620 may be sized and arranged to hold the reagent 510 within the structure via capillarity until the reagent 510 and containment structure 1620 are delivered. However the reagent 510 is held within the containment structure 1620 prior to delivery, upon delivery to the reaction site, the reagent 510 may be exposed to the products therein and thereby be release into the reaction.
[0108] In various exemplary embodiments, a plurality of containment structures 1620 may be adhered to the support 1605 in a format such that the containment structures 1620 are placed in conjunction with one or more rows of reaction chambers 805 in an arrayed sample holder, such as, for example, a microtiter plate.
[0109] In any of the exemplary embodiments of FIGS. 9-16, an air gap may be provided above the reagent 510 to the inner surface of the upper end portion 602 of the cap 601. The air gap may insulate the reagent 510 from heat during the expelling of the reagent 510 from the dispenser element 500 so as to minimize the risk of deactivating the reagent 510 if the reagent 510 is temperature-sensitive. Moreover, in various exemplary embodiments described with reference to FIGS. 9-15 above, a layer of oil may be provided in the dispenser element 500 above the reagent 510 to coat the inner surface so the dispenser and facilitate delivery of the reagent. In particular, such a layer of oil may be useful to facilitate delivery of relatively small volumes of liquid reagent by preventing capillary forces from causing the reagent to "stick" to the inner surfaces of the dispenser element 500. In that case, the oil may not be needed for the exemplary embodiment of FIGS. 14A-14E, as the reagent used in that embodiment may be in lyophilized form. An exemplary, non-limiting oil that may be used is silicone oil having a viscosity of about 5 centiStokes and in a volume ranging from about 20 microliters to about 50 microliters. In various exemplary embodiments, silicone oil having a viscosity ranging from about 0.5 centiStokes to about 5 centi Stokes and a boiling point ranging from about 100 0C to over about 250 0C may be used. In the various exemplary embodiments of FIGS. 9-15, it may not be necessary for the oil layer to be volatile at the operational temperatures of the particular assays being performed since delivery of the reagent is via mechanical mechanisms.
[01 10] Further, in various exemplary embodiments described herein, it may be desirable to keep the reagent delivery device relatively cool during a reaction prior to when it is desired to deliver the reagent. One exemplary mechanism to achieve such
cooling when using a thermal cycling device is to provide a cooled cover. FIG. 17A shows an exemplary embodiment of a thermal cycling device 1700 modified with a fan 1750 mounted over the cover 1735. Ventilation holes 1760 may be made through the cover 1735 so as to allow the fan 1750 to exhaust heat from underneath the cover 1735 out of the thermal cycling device 1700. In various exemplary embodiments, such as, for example, when performing a multi-stage reaction involving an initial PCR thermal cycling stage, it may be desirable to cool the cover 1735 to a temperature ranging from about 25 0C to about 40 0C to maintain the temperature of the reagent inside the caps at a temperature of less than about 50 0C, thereby minimizing the risk of rendering a temperature-sensitive reagent inactive and/or causing the reagent to be delivered too early in delivery devices that utilize heat as the mechanism by which to deliver the reagent. At the stage in the multi-stage reaction that it is desired to deliver the reagent, the fan 1750 may be turned off and the cover 1735 may be heated to reach a temperature sufficient to carry out remaining portions of a multi-stage reaction relying on thermal cycling and/or to release the reagent from the delivery device. For various exemplary embodiments, including, for example, for performing PCR/sequencing reactions, the cover 1735 may be heated to a temperature ranging from about 50 0C to about 105 0C, for example about 105 0C, after PCR has been completed.
[01 11 ] FIG. 17B depicts the thermal cycling device 1700 with the cover 1735 in an open position revealing a sample block 1730 which may be configured to receive, for example, a microtiter plate.
[01 12] Various exemplary embodiments of the present teachings also may include a thermal cycling device that is configured to be programmed so as to control
the temperature of both the sample block and the cover. In conventional thermal cycling devices, the sample block is controlled so as to cycle through a predetermined time-temperature program and the temperature of the cover is typically either heated to a single predetermined set temperature or left unheated. In various exemplary embodiments in accordance with the present teachings, however, it may be desirable to permit programming of both the cover and the sample block to permit various combinations of heating, cooling, and/or leaving unheated or uncooled those elements. In an exemplary embodiment, a controller may be used to program the thermal cycling device to perform PCR with the sample block cycling through various temperatures associated with a PCR cycle and with which those having ordinary skill in the art are familiar and the cover either left unheated or optionally, with the fan on, if desired), then to heat the cover (and. if previously on, turn the fan off) to a temperature sufficient to accomplish reagent delivery in accordance with various exemplary embodiments of the present teachings, and then control the temperature of the sample block in conjunction with optionally heating the cover or leaving the cover unheated to perform cycle sequencing. Of course, the preceding control over the cover temperature and the sample block is exemplary only and those ordinarily skilled in the art would recognize various other heating and/or cooling cycles for the sample block and the cover based on factors associated with a particular multi-stage reaction and/or reagent delivery being performed.
[01 13] FIGS. 18A and 18B show temperature profiles of the cover, the well of a microAmp™ Optical 96-well reaction plate (Applied Biosystems part number 4306737), and the inside of the cap (Applied Biosystems part number N801 1535), wherein a
thermal cycle was performed with the caps of a cap strip inserted into the wells of a microtiter plate. The wells were filled with 4 ul of PCR reaction mixture (primers (0.2 micromolars each), PCR master mix (1 x), and human genomic DNA (2 nanograms)) which was overlaid with 10 microliters of 5 centiStoke silicone oil. The PCR master mix used was AmpliTaq Gold® Fast PCR Mastermix, UP (Applied Biosystems part number 4390941 ). The temperature profiles in FIG. 18A correspond to a thermal cycle performed using a standard Veriti™ thermal cycler sold by Applied Biosystems wherein the cover remains unheated during the thermal cycle. The thermal cycle corresponding to the results shown in FIG. 18A was carried out using the following thermal cycle: 95 0C for 10 minutes; 35 cycles of 96 0C for 3 seconds, 62 0C for 3 seconds, and 68 0C for 8 seconds. As shown in FIG. 18A, the temperature inside the cap rises to 50 0C. The temperature profiles in FIG. 18B correspond to the same thermal cycle as for FIG. 18A, but using a Veriti™ thermal cycler that was modified by including a fan and ventilation holes to cool the cover and exhaust heat, as described with reference to the exemplary embodiment of FIG. 17. As shown in FIG. 18B, the temperature inside the cap remains below 4O0C during the thermal cycle. Maintaining the temperature below 40 0C may be useful when delivering a temperature-sensitive reagent, such as, for example, exonuclease I which degrades at about 50 0C. Maintaining the temperature of the cap at a relatively cool temperature may also be useful when using a cap made of a heat- shrinking and/or heat-softening material and/or when using a delivery mechanism that melts at a threshold temperature (e.g., an agarose or wax bead), in order to prevent premature reagent delivery.
[01 14] The following example of a PCR/sequencing reaction was performed using automated reagent delivery in accordance with the present teachings. EXAMPLE
[01 15] The PCR and sequencing reactions were performed in a microAmp™ Optical 96-well reaction plate (Applied Biosystems part number 4306737) using a Verti™ 96-WeII Thermal Cycler, 200 microliter format (Applied Biosystems part number 4375786). Six wells of the 96-well plate contained 4 ul of PCR reaction mixture (primers (0.2 micromolars each), PCR master mix (1 x), and human genomic DNA (2 nanograms)) which was overlaid with 10 microliters of 5 centiStoke silicone oil. The PCR master mix used was AmpliTaq Gold® Fast PCR Mastermix, UP (Applied Biosystems part number 4390941 ). Six caps on an eight-cap strip (Applied Biosystems part number N801 1535) were prepared. 15 microliters of 0.65 centiStoke silicone oil and 6 microliters of sequencing reagent (6 microliters containing BigDye Terminator v.3.1 (4 microliters), LGL019 primer (1 micromolar) and 2 units of exonuclease I) were added to each of the caps. A dispenser element, like the dispenser element of FIGS. 5A and 5B, made of Turcite® was inserted into each cap. The cap strip was inverted and sealed onto the corresponding six wells of the 96-well plate containing the PCR mix. A visible air gap in each cap (e.g., in the dome-shaped closed end 602 of each cap, as schematically depicted in the various exemplary embodiments shown herein) was observed.
[01 16] The fan over the cover of the thermal cycler was turned on and the heater for the cover was turned off. PCR was carried out using the following thermal cycle: 95 0C for 10 minutes; 35 cycles of 96 0C for 3 seconds, 62 0C for 3 seconds, and 68 0C for
8 seconds; and 4 0C for an indefinite time period (i.e., until the cycle sequencing profile is desired to be performed). After the PCR thermal cycle, the cover fan was turned off and the heater for the cover turned on to 105 0C. The reagent was observed to have been delivered to the wells. Exonuclease I digestion, exonuclease I heat-kill, and cycle sequencing was then carried out using the following thermal cycle: 37 0C for 15 minutes; 80 0C for 15 minutes; 96 0C for 1 minute; 25 cycles of 96 0C for 10 seconds, 50 0C for 5 seconds, and 60 0C for 75 seconds; and 4 0C for an indefinite time until the next processing step is desired.
[01 17] The sequenced samples were then processed with BigDye® Xterminator™ Purification kit (Applied Biosystems part number 4376486) with 45 microliters of SAM solution and 10 microliters of Xterminator™ beads added to each well and the microtiter plate was sealed with an 8-cap strip (Applied Biosystems part number N801 1535) and vortexed for 30 minutes at a speed sufficient to keep the beads suspended. The microtiter plate was centrifuged for 2 minutes at 100 rpm, after which the 8-cap strip was removed and the microtiter plate was loaded into an Applied Biosystems 373Ox/ DNA Analyzer. Five out of the six samples had clean sequence; one of the samples had a small amount of noise attributable to the reverse sequence (probably due to loss of exonuclease I activity). The result from one of the five clean sequencing runs is shown in FIG. 19.
[01 18] The following PCR primers were used in the Example: Fwd: 5'-TGTAAAACGACGGCCAGTAGCAGGACTCGTTCTCGCCC-S' Rev: 5'-CAGGAAACAGCTATGACCCCGGGACTGTCAAAGCCACA-S' [01 19] The LGL019 primer used in the Example was:
TGTAAAACGACGGCCAG*!, where * indicates phosphorothiate linkage. [0120] The sequence of amplicon used in the Example was:
5'-
TGTAAAACGACGGCCAGTAGCAGGACTCGTTCTCGCCCTAGAGTCTAACTGAATC
CCAAACTGGCAGTAAATGATGTCAGGGCTTCTGAGAGCATTCGCTTATCATCCAGT
GACCGGATGTGGAGTTAAGGGGATGGGCTGGACCCAAGGCAAGCCCCGACCATG
GCATGCCACTGAGCCACGAAGCTGGAGGCACCATACCTTTGCAGGTGAAACACTT
GATGTGGAAATGTTTGGTCTGGACCCGAAGCACTTCACCCTTGCAAGGCTCCCCA
CATTTATGGCAGTGAATGACAGGCTTCTCTGATGGGTGGTGAGGGTCCTGAGGGT
GGGCCACTGAAAGAAAATCAACAAAAGATCCAGGTGAGTAGAGAGCATTCCTGCA
AAAGGGGTCATCATGGTATCTCTGTAATTTTGGTGTCAAATGAGCCACAGAGATTG
ATTCGACAGTGTGAACAGAGCAGTCCCTCCAAGTCATAATCCTAGAGACACAAAGA
CCCATAAGACAGTTGTGGCTTTGACAGTCCCGGGGTCATAGCTGTTTCCTG-S'
[0121 ] Those having skill in the art would recognize that the various exemplary embodiments described herein may be modified to achieve reagent delivery for various multi-stage reactions aside from PCR/sequencing as set forth in many of the exemplary embodiments. Indeed, the various exemplary embodiments may be used to achieve automated reagent delivery for any of a variety of biochemical and/or chemical reactions, irrespective of whether or not those reactions have multiple stages or just a single stage for which reagent addition is desired.
[0122] The various exemplary embodiments of reagent delivery devices set forth herein may be configured to be disposable or may be configured for multiple uses with appropriate cleaning between uses. Moreover, in various exemplary embodiments, the reagent delivery devices may be provided to users in an empty form such that users can customize the reagent they want to add to the device for delivery. Alternatively, the reagent delivery devices in various exemplary embodiments may be prefilled with preselected reagent and/or volatile layer, if applicable, and provided to users with preselected reagents therein that may be used for predetermined reaction protocols.
[0123] Those having ordinary skill in the art would understand that features, components, steps, and/or materials described with respect to a particular exemplary embodiment set forth herein may be used with one or more other exemplary embodiments set forth herein and modifications made accordingly. It is to be understood that the particular examples and embodiments set forth herein are nonlimiting, and modifications to structure, dimensions, materials, and methodologies may be made without departing from the scope of the present teachings.
[0124] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the present teachings disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a scope being of a breadth indicated by the claims.
Claims
1. A method for delivering reagent during a thermal cycling reaction, the method comprising: placing a delivery device containing a reagent proximate to a reaction site; initiating a first stage of the reaction, wherein the first stage includes a period of an elevated temperature; and after completing the first stage of the reaction, dispensing the reagent from the delivery device to the reaction site, wherein the dispensing includes automatically actuating the delivery device to deliver the reagent.
2. The method of claim 1 , wherein the dispensing the reagent includes heating a portion of the delivery device to cause the reagent to be dispensed.
3. The method of claim 2, wherein heating the portion of the delivery device causes expansion of a substance within the portion of the delivery device.
4. The method of claim 3, wherein heating the portion of the delivery device causes expansion of a substance chosen from an expandable foam, nanoparticles, and a volatile substance.
5. The method of claim 1 , wherein dispensing the reagent from the delivery device comprises actuating a plunger to dispense the reagent from the delivery device.
6. The method of claim 1 , wherein the reagent is contained in a low-melt agarose gel and dispensing the reagent comprises melting the gel
7. A kit for sequencing nucleic acids, the kit comprising: a reagent comprising a nuclease and a nuclease-resistant sequencing primer; and a dispensing device holding the reagent, wherein the dispensing device is configured to automatically deliver the reagent to a reaction site.
8. The kit of claim 7, wherein the nuclease comprises exonuclease I.
9. The kit of claim 7, wherein the nuclease-resistant sequencing primer comprises at least one phosphorothioate linkage.
10. The kit of claim 7, wherein the dispensing device is configured to deliver the reagent upon at least a portion of the dispensing device reaching a predetermined temperature.
1 1. The kit of claim 7, wherein the dispensing device comprises a dispenser element defining a chamber holding the reagent.
12. The kit of claim 1 1 , wherein the chamber is configured to receive a substance that expands upon heating such that expansion of the substance in the chamber dispenses the reagent from the dispensing device.
13. The kit of claim 12, wherein the substance is chosen from an expandable foam, nanoparticles, and a volatile substance.
14. The kit of claim 7, further comprising a plunger configured to be automatically actuated to dispense the reagent from the delivery device.
15. The kit of claim 7, further comprising a low-melt agarose gel containing the reagent, wherein the low-melt agarose gel is configured to melt to delivery the reagent.
16. A method of sequencing nucleic acids, the method comprising: positioning a delivery device holding a reagent proximate a reaction site; amplifying nucleic acids at the reaction site in a first reaction to form amplified nucleic acids, wherein the first reaction comprises thermal cycling; after the amplifying step, automatically actuating the delivery device to deliver the reagent to the reaction site; and causing the amplified nucleic acids to react in a sequencing reaction with the reagent.
17. The method of claim 16, wherein the reagent comprises a nuclease and a nuclease-resistant sequencing primer.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US8146808P | 2008-07-17 | 2008-07-17 | |
US61/081,468 | 2008-07-17 | ||
US50458409A | 2009-07-16 | 2009-07-16 | |
US12/504,584 | 2009-07-16 |
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WO2010009426A2 true WO2010009426A2 (en) | 2010-01-21 |
WO2010009426A3 WO2010009426A3 (en) | 2010-04-01 |
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PCT/US2009/051041 WO2010009426A2 (en) | 2008-07-17 | 2009-07-17 | Devices and methods for reagent delivery |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016053881A1 (en) * | 2014-10-03 | 2016-04-07 | Life Technologies Corporation | Genetic sequence verification compositions, methods and kits |
EP3265232A4 (en) * | 2015-03-06 | 2018-08-15 | David James Saul | Method and device for preparing and extracting a biomolecule |
WO2024255603A1 (en) * | 2023-06-15 | 2024-12-19 | 深圳赛陆医疗科技有限公司 | Control method, gene sequencer, and storage medium |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7244559B2 (en) * | 1999-09-16 | 2007-07-17 | 454 Life Sciences Corporation | Method of sequencing a nucleic acid |
US20040038385A1 (en) * | 2002-08-26 | 2004-02-26 | Langlois Richard G. | System for autonomous monitoring of bioagents |
US20070166725A1 (en) * | 2006-01-18 | 2007-07-19 | The Regents Of The University Of California | Multiplexed diagnostic platform for point-of care pathogen detection |
AU2004209426B2 (en) * | 2003-01-29 | 2008-04-17 | 454 Life Sciences Corporation | Method for preparing single-stranded DNA libraries |
US20070117092A1 (en) * | 2003-04-08 | 2007-05-24 | Daksh Sadarangani | Dna analysis system |
US9278321B2 (en) * | 2006-09-06 | 2016-03-08 | Canon U.S. Life Sciences, Inc. | Chip and cartridge design configuration for performing micro-fluidic assays |
-
2009
- 2009-07-17 WO PCT/US2009/051041 patent/WO2010009426A2/en active Application Filing
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016053881A1 (en) * | 2014-10-03 | 2016-04-07 | Life Technologies Corporation | Genetic sequence verification compositions, methods and kits |
EP3265232A4 (en) * | 2015-03-06 | 2018-08-15 | David James Saul | Method and device for preparing and extracting a biomolecule |
WO2024255603A1 (en) * | 2023-06-15 | 2024-12-19 | 深圳赛陆医疗科技有限公司 | Control method, gene sequencer, and storage medium |
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WO2010009426A3 (en) | 2010-04-01 |
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