Patent Application
20060223069
DNA fragments generated during sequencing reactions may be subjected to size exclusion chromatography so as to reduce the amount of dye-labeled terminators loaded into an electrophoresis device. This can reduce dye artifacts that interfere with analysis of the sequencing reaction products. However, analysis of the products of many sequencing reactions, even those that are purified by size-based separation techniques, still suffer from dye artifacts.
Accordingly, there is a need for products and methods for reducing dye artifacts in chain extension reactions, e.g., in nucleic acid sequencing reactions.
Disclosed herein is the finding that contacting a chain extension reaction solution with a material that binds proteins, e.g., binds a DNA polymerase, will reduce dye artifacts when the products of the reaction are purified and analyzed.
Accordingly, one embodiment of the present invention provides is a method of reducing dye artifacts from a chain extension reaction, including (a) contacting a chain extension reaction solution including protein with at least one protein binding material to form a complex of the protein binding material with the protein; and (b) separating the complex from the chain extension reaction solution. In some embodiments of the invention, the protein is an enzyme, e.g., a DNA polymerase.
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
Disclosed herein is the finding that contacting a chain extension reaction solution with a material that binds proteins so as to create a complex of the protein-binding material with protein will reduce dye artifacts when the sample is purified so as to remove the complex and analyzed. Accordingly, in one embodiment the present invention provides a method of reducing dye artifacts from a chain extension reaction, including (a) contacting a chain extension reaction solution that includes protein with at least one protein binding material to form a complex of the protein binding material with the protein; and (b) separating the complex from the chain extension reaction solution.
In certain embodiments of the invention, the method further includes performing cycle sequencing prior to step (a). In certain embodiments of the invention, In certain embodiments of the invention, the method further includes purifying the chain extension reaction solution. In certain embodiments of the invention, the chain extension reaction solution is purified by size-based purification. In certain embodiments of the invention, the chain extension reaction solution is purified by size-based purification using a size-exclusion spin column. In certain embodiments of the invention, the chain extension reaction solution is purified by size-based purification using size-excluded ion exchange.
In certain embodiments of the invention, the method of claim 1, further includes analyzing the chain extension reaction solution on a fluorescent DNA sequencer.
In certain embodiments of the invention, the chain extension reaction solution is contacted with the protein binding material during the purification step. In certain embodiments of the invention, the chain extension reaction solution is contacted with the protein binding material prior to the purification step.
In certain embodiments of the invention, the protein is an enzyme. In certain embodiments of the invention, the enzyme is a DNA polymerase or RNA polymerase. In certain embodiments of the invention, the DNA polymerase is a thermostable DNA polymerase. In certain embodiments of the invention, the DNA polymerase is a Taq DNA polymerase. In certain embodiments of the invention, the Taq DNA polymerase is a mutated Taq DNA polymerase. In certain embodiments of the invention, the mutated Taq DNA polymerase is a mutated Taq DNA polymerase having a F667Y mutation. In certain embodiments of the invention, the protein is associated with a dye-labeled dideoxynucleotide.
In certain embodiments of the invention, the chain extension reaction is a DNA sequencing reaction. In certain embodiments of the invention, the protein binding material is a membrane. In certain embodiments of the invention, the membrane is a porous membrane. In certain embodiments of the invention, the porous membrane includes pores ranging from about 0.01 μm to about 200 μm in size. In certain embodiments of the invention, the porous membrane includes pores ranging from about 0.05 μm to about 100 μm in size. In certain embodiments of the invention, the porous membrane includes pores ranging from about 0.1 μm to about 50 μm in size. In certain embodiments of the invention, the porous membrane has a thickness ranging from about 0.1 μm to about 2000 μm. In certain embodiments of the invention, the porous membrane has a thickness ranging from about 0.5 μm to about 1500 μm. In certain embodiments of the invention, the porous membrane has a thickness ranging from about 0.5 μm to about 1000 μm.
In certain embodiments of the invention, the protein binding material is a solid support. In certain embodiments of the invention, the solid support is a microsphere. In certain embodiments of the invention, the microsphere is a polystyrene microsphere. In certain embodiments of the invention, the microsphere is a polymer microsphere having a diameter from about 100 nm to about 2000 μm. In certain embodiments of the invention, the microsphere has a diameter from about 500 nm to about 1000 μm. In certain embodiments of the invention, the microsphere has a diameter from about 1 μm to about 200 μm. In certain embodiments of the invention, the microsphere is a nonporous microsphere. In certain embodiments of the invention, the microsphere is a microporous microsphere. In certain embodiments of the invention, the microsphere is a macroporous microsphere. In certain embodiments of the invention, the microsphere is a magaporous microsphere.
In certain embodiments of the invention, the protein binding material forms a layer on the surface of a solid support. In certain embodiments of the invention, the solid support is a porous membrane. In certain embodiments of the invention, the protein binding material is associated with the solid support via covalent bonds.
In certain embodiments of the invention, the protein binding material is mixed with a size-based purification media.
In certain embodiments of the invention, the protein binding material is layered on a size-exclusion spin column.
In certain embodiments of the invention, the protein binding material is contacted with the chain extension reaction solution prior to chain extension reaction. In certain embodiments of the invention, the protein binding material remains in contact with the chain extension reaction solution during chain extension reaction.
Certain embodiments of the invention also provide a microfluidic device including a first region that includes a protein binding material capable of binding with a protein in a chain extension reaction solution.
Certain embodiments of the invention also provide a spin column including protein binding material capable of binding with a protein in a chain extension reaction solution.
In certain embodiments of the invention, proteins are removed from a solution, e.g., proteins complexed with dye-labeled nucleotides, from a solution so as to reduce the generation of dye artifacts when the solution is analyzed. The dye-labeled nucleotide is a dye-labeled nucleotide that has not been incorporated into a strand of polynucleotides during the chain extension reaction, i.e., an unincorporated dye-labeled nucleotide.
Certain embodiments of the invention provide a spin column that includes a chain extension reaction product that has been treated so as to remove unincorporated terminators.
Certain embodiments of the invention provide a spin column that includes a chain extension reaction product that has been treated so as to remove proteins.
Chain extension reactions include reactions involving the synthesis of a strand of polynucleotides, e.g., a strand of DNA. These reactions can involve a template-directed polynucleotide extension processes using at least one polymerase. Dye-labeled nucleotides and/or polynucleotides may be utilized in the chain extension reactions. Chain extension reactions include, but are not limited to, polymerase chain reactions, sequencing reactions, single base extension (SBE) reactions, mini-sequencing reactions, and the like. A chain extension reaction solution is the solution in which a chain extension reaction takes place.
A protein binding material is a material with which proteins can bind. While certain protein binding materials are exemplified herein, the invention also is meant to encompass other materials that bind with proteins so as to allow the removal of protein from a solution, e.g., from a chain extension reaction solution.
The protein present in the chain extension reaction can be any protein, e.g., any protein that one of skill in the art might add to the chain extension reaction, including proteins that are present as impurities. In some embodiments of the invention, the protein is an enzyme. In some embodiments of the invention, the enzyme is an RNA polymerase or a DNA polymerase, e.g., a Taq DNA polymerase. The Taq DNA polymerase may be a mutated Taq DNA polymerase, e.g., a Taq DNA polymerase having a F667Y mutation. The DNA polymerase may or may not be a thermostable DNA polymerase. The DNA polymerase may have a naturally occurring amino acid sequence or may be a mutant derived in part from a naturally occurring amino acid sequence.
Polymerases include, but are not limited to the polymerases disclosed in U.S. Pat. Nos. 6,534,269; 6,399,304; 6,365,350; 6,265,193; 6,197,555; 5,876,934; 5,871,929; 5,466,591; and 6,228,628 and in Abramson, “Thermostable DNA Polymerases: An Update”, In PCR Applications: Protocols for Functional Genomics, Eds. M. A. Innis, D. H. Gelfand, and J. J. Sninsky, Academic Press, 33-47 (1999); Ignatov et al., FEBS Letters, 448, 145-148 (1999); Ignatov et al., FEBS Letters, 425, 249-250 (1998); Kalman et al., “Thermostable DNA Polymerases with Altered Discrimination Properties”, Genome Science and Technology, 1, Abstract A-14, P-42 (1995); and Tabor et al., PNAS, 92, 6339-6343 (1995). In some embodiments, the protein is a polymerase having reverse transcriptase activity. In some embodiments, the protein is a DNA dependent DNA polymerase. In some embodiments of the invention, the protein is associated with a dye-labeled dideoxynucleotide. In some embodiments of the invention, the chain extension reaction is a DNA sequencing reaction.
In some embodiments of the invention, the protein binding material is a solid support, a filter, or a membrane. In some embodiments of the invention, the protein binding material is mixed with a size-based purification media. In some embodiments of the invention, the protein-binding material is coated onto the surface of a sized-based purification medium. In some embodiments of the invention, the protein binding material is layered on a size-exclusion spin column.
In some embodiments of the invention, the method further includes performing cycle sequencing prior to contacting the chain extension reaction solution with the protein binding material. In some embodiments of the invention, the method further includes purifying the chain extension reaction solution, e.g., by size-based purification. In some embodiments of the invention, the chain extension reaction solution is purified by size-based purification using a size-exclusion spin column. In some embodiments of the invention, the chain extension reaction solution is purified by size-based purification using size-excluded ion exchange. In some embodiments of the invention, the chain extension reaction solution is contacted with the protein binding material during the size-based purification step. In some embodiments of the invention, the chain extension reaction solution is contacted with the protein binding material prior to the size-based purification step. In some embodiments of the invention, the method further includes analyzing the chain extension reaction solution on a fluorescent DNA sequencer.
Another embodiment of the invention is a kit for the purification of the sequencing reaction products. Kits of the invention may include a protein binding material and size-exclusion chromatography material. Kits of the invention may further include one or more reagents for performing a nucleic acid sequencing reaction. Such reagents include, but are not limited to a dye labeled terminators, primers, and/or DNA polymerases. The kits may also include instructions for their use for purifying sequencing reaction products.
Dye-labeled terminators may be removed from sequencing reaction products when sequencing nucleic acid sequences, such as DNA. For example, following cycle sequencing (e.g., Sanger sequencing) a purification step can be performed in an attempt to remove reaction components that could interfere with the analysis of the sequencing reaction products. In the case of cycle sequencing with dye-labeled dideoxynucleotides (i.e., dye terminators), it is helpful to remove the dye-labeled dideoxynucleotides that have not been incorporated into the DNA sequencing ladders. Failure to remove these unincorporated dye-labeled dideoxynucleotides results in artifacts in the electropherogram when the sample is analyzed on a fluorescent DNA sequencer. These artifacts, referred to herein as “dye artifacts” or simply as “artifacts”, can interfere with the accurate identification of the nucleic acid sequence.
While not a limitation of the present invention, it is believed that an interaction between a dye-labeled dideoxynucleotide and a DNA polymerase used in a sequencing reaction leads to the formation of a dye artifact observed following size-based purification procedures. The polymerase is believed to form a relatively high molecular weight complex with a dye-labeled dideoxynucleotide. This complex is thought to be large enough and stable enough to pass unretained through the spin column and to elute with the DNA. However, it appears that when this complex is subjected to electrophoretic injection conditions, the complex disassociates and the dye-labeled dideoxynucleotide can be observed as a dye artifact. Based on this finding, methods and products for reducing, e.g., eliminating, dye artifacts from sequencing reactions by removing the complex prior to analysis have been developed.
The invention described herein enables the use of a microfluidic platform to perform reactions, e.g., chain extension reactions. Such platforms are known to the art worker, e.g., described in U.S. Pat. Nos. 6,613,525; 6,582,987; 6,344,326; 6,103,537; and 6,074,827. Such platforms can use size-excluded ion exchange (SEIE) as a purification method for sequencing reactions, and thus can suffer from the same problems as other size-based purifications. SEIE is described, e.g., in U.S. Patent Application Publication Numbers 20040055956; 20040018559; 20040018116; 20040016702; and 20030228706. By incorporating the methods of the present invention with the methods utilizing a microfluidic platform, these problems can be avoided.
Thus, certain embodiments of the invention provide devices, e.g., microfluidic devices, that are capable of being used to carry out a method of the invention, e.g., to remove proteins with a protein binding material, e.g., from a solution. For example, certain embodiments of the invention provide a microfluidic device having a region including a protein binding material. Certain embodiments of the invention provide a microfluidic device having a first region that includes a protein binding material and a second region for removing unincorporated nucleotide, e.g., dye-labeled terminators. The first and second regions can be connected to each other by a channel.
In some embodiments, the invention involves the use of a protein binding material that is added before, during and/or after a chain extension reaction. Such a material can bind proteins, e.g., DNA polymerases, and in doing so reduces or prevents the formation of dye artifacts when using purification methods, e.g., size-based purification methods, prior to fluorescent DNA sequencing.
It was discovered that if sequencing reaction products were reacted with an protein-binding support prior to or during a size-based purification, the dye artifacts were reduced. In some embodiments of the invention, the sequencing reaction products can be filtered through an protein-binding membrane such as the Micropure-EZ (Millipore, Billerica, Mass., USA), prior to purification using a spin column. This reduces dye artifacts in the electropherogram. (
Another embodiment of the invention includes methods that include the step of mixing the protein binding material with size-based purification media. For example, HiTrap Phenyl Sepharose 6 can be mixed with the size-based purification media (e.g., 100 uL HiTrap Phenyl Sepharose 6 can be mixed into an 800 uL CentriSep column) to cause reduction of the dye artifacts. (
Another embodiment of this invention involves coating the protein binding material onto the surface of a size-based purification medium, e.g., a porous anion exchange resin coated with multilayers of polyelectrolytes with alternative charges. (see U.S. patent application Ser. No. 10/780,963).
Another embodiment of this invention involves layering the protein binding material on top of a size-exclusion spin column. For example, 100 uL HiTrap Phenyl Sepharose 6 can be pipetted onto the top of an 800 uL CentriSep column (Princeton Separations, Adelphia, N.J., USA) for reducing the dye artifacts. (
These are only examples of materials (e.g. membranes and chromatography supports) that can bind proteins, e.g. DNA polymerases, and thus reduce dye artifacts from sequencing reactions. Any material that is capable of binding a protein, e.g., a material capable of binding a DNA polymerase, may also be used. These materials include, but are not limited to, POROS 20 HP2 (Applied Biosystems, Foster City, Calif., USA), HiTrap Octyl FF, HiTrap Butyl FF, HiTrap Phenyl Sepharose 6 FF-high sub, Octyl Sepharose and Butyl Sepharose (all from AP Biotech), poly(4-vinly phenol), MW 1500-7000 (Polysciences, Inc, Warrington, Pa., USA), or Isolute ENV+ (Argonaut Technologies, Foster City, Calif., USA).
Spin Columns
Spin columns, and methods for making spin columns, are known to the art worker. For example, several companies manufacture and sell spin columns, including Bio-Rad (Hercules, Calif., USA), Princeton Separations (Adelphia, N.J., USA), Amersham Pharmacia Biotech (Piscataway, N.J., USA), and Millipore Corporation (Billerica, Mass., USA). Spin columns may also be easily prepared by those of skill in the art, and many variations of procedures can be used, including, for example, filling empty columns with a size exclusion chromatography resin. Examples of useful resins for self-filled columns are Sephadex G-50 (Amersham Pharmacia Biotech, Piscataway, N.J., USA), Bio-Gel P-10 and Bio-Gel P-30 (Bio-Rad, Hercules, Calif., USA). While these products may be used in removing some unincorporated dye artifacts from DNA sequencing reactions, they suffer from residual dye artifacts when used with modern DNA sequencing kits such as BigDye Terminators v.3.1 (Applied Biosystems, Foster City, Calif., USA).
Thus, certain embodiments of the invention provide devices, e.g., spin columns, that are capable of being used to carry out a method of the invention, e.g., to remove proteins with a protein binding material, e.g., from a solution.
Solid Supports as Protein-Binding Materials
In some embodiments, this invention relates to the use of solid supports to remove proteins, e.g., DNA polymerases, from sequencing reactions, thereby reducing, if not eliminating, dye artifacts. The solid supports may be porous membranes having pore sizes ranging from about 0.01 to about 200 μm, e.g., from about 0.05 to about 100 μm, e.g., from about 0.1 to about 50 μm, and thicknesses ranging from about 0.1 to about 2000 μm, e.g., from about 0.5 to about 1500 μm, e.g., from about 0.5 to about 1000 μm. The solid supports may be polymer microspheres having particles size ranging from about 100 nm to about 2000 μm, e.g., about 500 nm to about 1000 μm; e.g., about 1 to about 200 μm. The polymer microspheres may be microporous (pore size less than about 30 Å), macroporous (pore size about 30-4000 Å), or magaporous (pore size greater than about 4000 Å, e.g., Magnapore™ prepared by Polygenetics, Los Gatos, Calif., using the high-internal-phase emulsion (HIPE) technology (see U.S. Pat. Nos. 4,522,953 and 5,583,162). In some embodiments, the pore sizes can range from about 10 Å to about 10,000 Å, e.g., from about 100 Å to about 8,000 Å. In some embodiments, the pore sizes can be greater than about 10,000 Å.
In the case of porous membranes, the analyte can be allowed to permeate (i.e., filter) through the membrane pores allowing the protein-dye terminator complex to be sequestered. In the case of polymer microspheres, the purification can be in batch or chromatography mode by exposing the analyte to a batch of polymer microspheres, or by eluting through a column of packed microspheres, respectively, allowing the protein-dye terminator complex to be sequestered. In some embodiments, surfaces may be non-porous microspheres, non-porous membranes, or other non-porous surfaces which are prepared e.g., surface functionalized, to present a protein binding surface.
Solid Supports Containing a Phenolic Moiety
One embodiment of the invention involves the removal of dye artifacts by polymer solid supports 2 containing phenolic groups on their surface, as illustrated in Scheme 1. For example, the solid support may be polymer microspheres 1 prepared by free radical polymerization of styrene and divinylbenzene, and subsequent hydroxylation. (Scheme 1) Those of ordinary skill in the art can conduct nitration/amination on 1 and subsequent diazotization/hydrolysis to prepare 2 with phenolic groups on the surface. A porogen, for example, hexanol, can be added into the monomers during emulsion polymerization to give porous polystyrene microspheres. Those of ordinary skill in the art can adjust the porosity and pore size by tailoring the porogen concentration and experimental conditions. A commercially available product with a chemical composition similar to 2 is Isolute ENV+. (Argonaut, Foster City, Calif.)
Polymer microspheres containing phenolic moiety can also be prepared by emulsion copolymerization of a hydroxystyrene precursor with at least one comonomer, which could be water-insoluble or slightly water-soluble, and a crosslinker as illustrated in Scheme 2. A porogen can also be added to tailor the porosity. In Scheme 2, the hydroxystyrene precursor is, e.g., acetoxystyrene 3 that hydrolyzes to give phenolic moiety in a subsequent step. The comonomer can be styrene; (meth)acrylamides, e.g., N-ethyl(meth)acrylamide, N-methyl (meth)acrylamide, N,N-dimethyl(meth)acrylamide, and N-butyl(meth)acrylamide; (meth)acrylates, e.g., methyl(meth)acrylate, ethyl(meth)acrylate, 2-ethylhexyl (meth)acrylate; N-vinylamides, e.g., N-methy-N-vinyl acetamide; vinyl alkyl ethers, e.g., vinyl methyl ether and vinyl butyl ether; ally alkyl ethers, e.g., allyl ethyl ether, allyl butyl ether, and allyl benzyl ether; or a combination two or more thereof. The crosslinker includes but is not limited, e.g., to divinylbenzene, N,N′-methylenebisacrylamide, ethylene diacrylate, pentaerythritol triacryalte, or tetraethylene glycol diacrylate. Those of skill in the art can determine the appropriate amount of crosslinker to be used to prepare polymer microspheres having the desired physical and chemical properties. This phenolic polymer can also be used to fabricate porous membranes. Another protocol for the preparation of polystyrene microspheres containing phenolic moiety is reported by Jean M. J. Frechet et al., in Polymer, 20, 675-680 (1979).
Solid Supports Based on Polyvinylidene Difluoride (PVDF)
Another embodiment of the invention involves the use of PVDF, or its derivatives, to reduce dye artifacts by removing the protein-dye terminator complex. PVDF in particle or membrane form can be used to reduce dye artifacts. Ion-exchange membranes prepared from sulfonation of PVDF films can also be used to bind complexes of the protein binding material and the protein. (Scheme 3; Richards et al., 1964 and Yang et al., 2003) The presence of negatively charged sulfonate groups reduces passive adsorption of DNA fragments.
Solid Supports Based on Crosslinked Dextran and Agarose
Some embodiments of the present invention involve the use of crosslinked, alkylated or arylated dextran and agarose to bind the protein-dye terminator complex. Crosslinked dextran, includes but not limited to, Sephadex® (Scheme 4) or Sephacryl® (Scheme 5), and crosslinked agarose includes but not limited to Sepharose®. (Scheme 6)
The surface hydrophilicity of rigid, highly crosslinked dextran and agarose beads or porous membranes can be tailored, for example, by introducing alkyl, aryl, or alkylaryl groups via the hydroxyl moiety. For example, a butyl or phenyl group can be covalently bonded onto the surface by reacting the surface hydroxyl groups with epichlorohydrin (see Scheme 7) and a subsequent reaction with an alkoxide or phenoxide. Due to various strength of hydrophobic interaction, proteins, peptides and enzymes will be bound to the surface. The hydrophobic groups (R in Scheme 7) on the surface bind to peptides, proteins, enzymes and protein-dye terminator complexes due to hydrophobic interaction, whereas the neutral, hydrophilic matrix minimizes passive adsorption of negatively charged DNA fragments.
Solid Supports Based on Highly Crosslinked Hydrogels
One embodiment of the invention provides for the removal of protein-dye terminator complexes with highly crosslinked hydrogels containing hydrophobic moieties on its surfaces. The hydrophobic moiety contributes to the hydrophobic interaction for binding protein-dye terminator complexes, whereas the hydrophilic matrix of the hydrogel reduces passive adsorption of DNA fragments. The hydrogel can be prepared by polymerizing a water-soluble monomer, at least one water-soluble, slightly water-soluble, or water-insoluble co-monomer, and a crosslinker. The crosslinker can be water-soluble, slightly water-soluble, or water-insoluble, e.g., N,N′-methylenebisacrylamide, tetraethyene glycol di(methy)acrylate, or pentaerythritol tri(meth)acrylate. Desired physical and chemical properties of the hydrogen can be obtained by choosing the appropriate concentration and chemical structure of the crosslinker. In various embodiments of the invention, the co-monomer includes a hydrophobic moiety, e.g., butyl(methy)acrylate, styrene, 2-ethylhexy (meth)acrylate, butyl(meth)acrylamide, N-butyl-N-vinyl formamide, N-butyl-N-vinyl-acetamide, N-phenyl-N-vinyl acetamide, vinyl methyl ether, vinyl butyl ether, allyl ethyl ether, allyl butyl ether, allyl benzyl ether, or a combination thereof. In various embodiments of the invention, the co-monomer includes an anionic moiety, e.g., acrylic acid, methacrylic acid, vinyl sulfonic acid, or styrene sulfonic acid. The anionic moiety of the hydrogel matrix can reduce passive adsorption of negatively charge DNA fragments, whereas the hydrophobic moiety can bind to protein-dye terminator complexes via hydrophobic interaction. Normal emulsion polymerization, inverse emulsion polymerization, membrane emulsification/polymerization techniques can be used to prepared hydrogel particles. Desirable particle size can be obtained by tailoring the conditions for emulsification and the use of appropriate surfactant.
In some embodiments of the invention, finished sequencing reaction products, e.g., products from a sequencing reaction following thermal cycling, can be added to a vessel containing a protein binding material prior to purification of the reaction. In some embodiments of the invention, the protein binding material can be added directly to the sequencing reaction prior to thermal cycling the reaction. The protein binding material can remain in the same vessel with the sequencing reaction while the reaction is thermal cycled.
In some embodiments of the invention, the protein binding material may be a bead or particle such as a chromatography support, or insoluble particles such as polymer microspheres. The protein binding material should be capable of binding a protein, e.g., a DNA polymerase, in the reaction buffer at room temperature, but not interfere with the reaction under the thermal cycling conditions.
The invention will now be illustrated by the following non-limiting Examples.
It was discovered that if sequencing reaction products were contacted with a material that binds proteins prior to or during size-based purification, dye artifacts were not observed when the products were analyzed.
DNA Sequencing Reactions
20 uL sequencing reactions were performed using 8 uL of BigDye terminator (BDT) v.3.1 ready reaction mix for each 20 uL reaction. The sequencing primer was 3.2 pmol of M13 reverse primer per reaction. (5′-CAGGAAACAGCTATGACC-3′; SEQ ID NO:1) The template was a PCR product with universal M13 priming sites.
Approximately 150 ng of the template was used per 20 uL reaction. The reactions were cycled in a GeneAmp 9700 (Applied Biosystems, Foster City, Calif., UA) using default sequencing conditions (96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 240 seconds; 25 times) and pooled. The PCR product that was sequenced in
Enzyme Removal
An aliquot of 50 uL of pooled sequencing reaction products was added to the Micropure-EZ Enzyme device and spun at 13,000×g on a bench top centrifuge for 30 seconds. The liquid that passed through the membrane was then purified with either CentriSep or SEIE. In the case of purification using HiTrap resin prior to CentriSep columns (
CentriSep Purification
CentriSep columns from Princeton Separations were hydrated with 800 uL of high purity deionized water for 2 hours. An aliquot of 20 uL of sequencing reaction products was eluted through the columns. The purified samples were transferred to the ABI 3100 for analysis.
Mixed Affinity/CentriSep Columns
A CentriSep column was hydrated with 800 uL of water and vortexed. The column was allowed to stand for 30 minutes. An aliquot of 100 uL HiTrap resin slurry was added with a pipette and was vortexed. Other affinity resins can also be used. The column was then processed per the instruction included with the CentriSep columns.
Layered Affinity/CentriSep Columns
A CentriSep column was hydrated with 800 uL of water and vortexed. The column was allowed to stand for 30 minutes. Following the 30 minutes hydration, the spin column was uncapped and allowed to drain by gravity for about 10 minutes. An aliquot of 100 uL HiTrap resin slurry was carefully pipetted onto the top of the CentriSep resin bed. (Other affinity resins may also be used.) The column was then processed as per the instruction included with the CentriSep columns.
Size-Excluded Ion Exchange Purification
An aliquot of 1 uL of slurry (see 20040018559A1; 20040016702A1; and U.S. patent application Ser. No. 10/780,963 for a full description of this material) was transferred to a MicroAmp tube. An aliquot of 1 uL of sample was added, and the mixture was pipette-mixed, then vortexed for 1 minute. To the mixture, 6 uL of deionized water was added, vortexed, the resin spun down to the bottom of the tube, and the supernatant transferred to the ABI 3100 for analysis.
Electrophoresis Conditions
Electrophoresis was run on an ABI 3100 with a 36 cm capillary array, POP-6 polymer and a modified version of the default “rapid seq” method. The method was modified by reducing the injection conditions from 1000 V for 22 seconds to 500 V for 11 seconds. Injections were made from deionized water.
This Example describes experiments indicating that the enzyme binding materials can be added directly to the sequencing reaction prior to thermal cycling. The enzyme binding material can thus remain in the same vessel with the sequencing reaction while the reaction is thermal cycled. The enzyme binding material should be capable of binding a protein, e.g., a polymerase, in the reaction buffer at room temperature, but the enzyme binding material should not interfere with the reaction under the thermal cycling conditions.
A BigDye v.3.1 terminator reaction solution was prepared by combining 80 uL of BigDye Terminator v. 3.1 Ready Reaction Mix (Applied Biosystems) with 10 uL of M13 universal forward sequencing primer (5′-TGTAAAACGACGGCCAGT-3′; SEQ ID NO:2) at 3.2 pmol/uL, 15 uL of pGEM (200 ng/uL) plasmid template, and 95 uL of deionized water.
Aliquots of 20 uL of the reaction solution were pipetted into 6 wells of a 96-well PCR tray. Two different types of protein-binding materials were tested as additives to the reaction. Both of the additives described below (100 uL each) were washed five times with 500 uL aliquots of deionized water prior to addition to the reaction solution. After washing, all materials were spun down and left with minimal water above the level of the resin.
The solutions were prepared were as follows. In tubes 1 & 2, 20 uL of reaction solution with no additives was used as a control. In tubes 3 & 4, 20 uL of reaction solution was combined with 1 uL POROS 20 HP2 (Applied Biosystems, Foster City, Calif., USA). In tubes 5 & 6, 20 uL of reaction solution was combined with 1 uL Phenyl Sepharose 6 FF high sub (Amersham Biosciences, Uppsala, Sweden).
The PCR tray was then thermal cycled using the default BigDye cycling conditions on an Applied Biosystems 9700 thermal cycler. Following thermal cycling, the 96 well tray was removed from the thermal cycler and was vortexed for 1 minute. The plate was then spun down in a centrifuge.
Six CentriSep columns were hydrated with 800 uL deionized water, vortexed, and allowed to stand for 2 hours, as per the manufacturer's instructions. Prior to loading the CentriSep columns, the rehydrated columns were centrifuged at 3000 rpm for 2 minutes in a bench top centrifuge. Aliquots of 20 ul of each sequencing reaction solution were pipetted onto a CentriSep column. No special care was taken to avoid pipetting enzyme binding resin along with the sample; in fact some resin was transferred to the CentriSep column. The columns containing samples were centrifuged at 3000 rpm for 2 minutes in a bench top centrifuge. Approximately 20 uL of sample was recovered from each column. An aliquot of 20 uL purified sample was transferred to a 96-well plate for analysis on a DNA sequencer. The samples were analyzed on an ABI 3100 DNA sequencer (Applied Biosystems) using a 36 cm capillary array using POP-6 polymer and the default RapidSeq36 method.
The results are shown as
While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.
All publications, patents and patent applications cited herein are herein incorporated by reference.
