Patent Application
20110195477
This application relates to microcapsules comprising an enzyme and nucleic acid template encapsulated in a semipermeable membrane. Microcapsules of the invention can be used in enzyme-mediated reactions, for instance, DNA amplification and sequencing.
DNA sequencing encompasses biochemical methods for determining the order of nucleotide bases (adenine, guanine, cytosine and thymine/uracil) in a nucleic acid. Sanger sequencing, otherwise known as dideoxynucleotide sequencing or chain-termination sequencing, was developed in the mid-1970s and continues to be widely used in DNA sequencing. The Sanger sequencing method utilizes the incorporation of 2′, 3′-dideoxynucleotide triphosphates (ddNTPs) in a growing DNA chain. To perform a Sanger sequencing reaction, a single stranded DNA template, a primer, polymerase, deoxynucleotide triphosphates (dNTPs) and one or more ddNTPs are mixed together and incubated under conditions that allow for primers to anneal to the single stranded DNA and elongate to form a complementary DNA strand. Either dNTPs, primer or ddNTPs are labeled in the reaction. During elongation, both dNTPs and ddNTPs (usually at a concentration of about 1% total concentration of dNTPs) are incorporated in the complementary DNA strand. Unlike dNTPs, ddNTPs lack a 3′-OH group and are unable to form a phosphodiester bond with the next deoxynucleotide. As a result, the DNA chain is terminated at the point of incorporation of the ddNTP. The sequence of the DNA is determined by separating the resulting labeled DNA fragments by electrophoresis. See, Sanger, F. and Coulson, A. R., 1975, J. Mol. Biol. 94: 441-448; Sanger, F. et al., 1977, Nature. 265(5596): 687-695; and Sanger, F. et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 75: 5463-5467.
Despite being a commonly-used method, Sanger sequencing is expensive, labor intensive and not well suited for high-throughput sequencing. As a result, several amplification and sequencing technologies have been developed in attempts to overcome the drawbacks associated with Sanger sequencing. Amplification technologies developed to be used in conjunction with sequencing include, for instance, emulsion PCR and bridge PCR. New sequencing technologies include, for instance, pyrophosphate sequencing, base-at-a-time sequencing by synthesis, and supported oligo ligation detection. However, the read lengths produced by many of these newer technologies tend to be shorter than those produced by Sanger sequencing and the raw accuracy of the reads tend to be lower.
A major problem that has faced researchers has been the inability to prepare, in a short period of time and at low cost, the numerous nucleic acid samples necessary for large-scale sequencing. Sanger sequencing, as well as many other sequencing methods, requires amplified nucleic acid template as starting material. Methods typically used for amplifying nucleic acid template for subsequent sequencing such as cloning and random-primed PCR are ill-suited for large scale sequencing. Cloning is labor intensive and, as a result, both time consuming and costly. Cloning biases can also occur, which may prevent some nucleic acid regions from being represented. Although random-primed PCR is not as laborious as cloning and can be engineered to amplify a plurality of nucleic acids in a single reaction, the method is not preferred because the amplified nucleic acids are usually not representative of the starting nucleic acid templates (i.e., amplification bias occurs).
Emulsion PCR is an example of an amplification method that has been developed to amplify nucleic acids for large-scale sequencing. In emulsion PCR, a nucleic acid template, primer coated bead, and amplification solution are suspended in a heat stable water-in-oil emulsion to form thousands to millions of separate microreactors. Each microreactor optimally contains a single nucleic acid template and a single bead. Within each microreactor, nucleic acid amplification takes place on the bead and amplified nucleic acid fragments remain attached to the bead. By placing thousands to millions of microreactors in a single tube and subjecting the tube to conditions necessary for PCR, thousands to millions of nucleic acid templates can be amplified in parallel without competition among the reactions. Sequencing of amplified nucleic acid fragments can subsequently be performed by breaking the microreactors and isolating the beads containing amplified nucleic acid fragments. See, e.g, U.S. Patent Application 200500079510;Margulies, M. et al., 2005, Nature. 437: 376-380; Shendure, J. et al., 2005, Science. 309: 1728-1732; Williams, R. et al., 2006, Nature Methods. 3(7): 545-550; and Wicker, T. et al., 2006, BMC Genomics. 7: 275.
Sanger sequencing relies on electrophoresis methods to “read” a nucleic acid sequence after a sequencing reaction has been performed. Electrophoresis methods separate nucleic acid fragments based on size so that fragments differing by only one nucleotide can be resolved. Most genomic sequencing strategies to date have relied on capillary array electrophoresis (CAE). For instance, CAE was used by the Celera human sequencing project to sequence cloned nucleic acid fragments, which were then assembled electronically (Fredlake, C. et al., 2006, Electrophoresis. 27: 3689-3702). Commercial CAE instruments are able to produce raw sequencing reads of about 650-700 phred 20 (Q20) quality bases per capillary in about 1-2 hours with 100-400 capillaries per instrument. Id.
Other electrophoretic methods and non-electrophoretic methods have been developed for use with sequencing, including, but not limited to, microfluidic devices such as on-chip sequencing systems and massively parallel sequencing systems for use with sequencing-by-synthesis methods. Reports of read lengths produced by microfluidic devices varies greatly from about 320 bases to over 800 bases depending on sequencing system used. Massively parallel sequencing systems, for instance, 454 Life Sciences' GS-20, which uses an enzymatic sequencing-by-synthesis technique known as pyrosequencing, reportedly averages a read length of 100 bases and can read up to 25 million bases in one 4-hour run. Id. Although the GS-40 seems well suited for sequencing small genomes such as bacterial and viral genomes, the shorter read lengths obtained with this system relative to Sanger sequencing make this system less suitable for sequencing larger genomes at present.
The present invention provides materials and methods that enable massively-parallel sample preparation and nucleic acid sequencing in a single reaction vessel. The invention allows single-vessel, batch processing of samples to produce sequence for high-depth coverage of genomic fragments as well as large genomes. Using compartmentalized sequencing vesicles as taught herein, a user is able to conduct single molecule amplification and Sanger sequencing reactions in the same compartment with free exchange of reagents and by-products, but not large macromolecules such as nucleic acids and polymerase.
Thus, according to the invention, sample preparation and sequencing are conducted in a semi-closed environment in which one can produce a clonal population of the nucleic acids to be sequenced. In a preferred embodiment, the vesicles are semi-permeable membrane droplets housing an aqueous compartment for conducting DNA amplification and sequencing reactions. Use of the invention allows one to conduct massively-parallel sequencing reactions, especially Sanger-type reactions, in a self-contained environment.
Microcapsules of the invention comprise a semipermeable membrane containing one or more enzymes and a nucleic acid template for use in an enzyme-mediated reaction. The semipermeable membrane allows low molecular weight molecules to cross the membrane while preventing large molecular weight molecules from crossing the membrane. The effect is that the enzymes and nucleic acid template are unable to exit the aqueous core of the microcapsule by simple diffusion.
In a preferred embodiment, each microcapsule comprises a semipermeable membrane surrounding one or more polymerase enzymes and a nucleic acid template. Microcapsules are used to amplify a nucleic acid template by rolling circle amplification (RCA) or polymerase chain reaction (PCR) and subsequently sequence the amplified nucleic acid template. Microcapsules are used to sequence an amplified nucleic acid template using Sanger sequencing biochemistry but on a larger scale and without the labor-intensive sample preparation steps generally associated with Sanger sequencing.
A plurality of microcapsules can be used to amplify thousands, even millions or hundreds of millions, of nucleic acid templates simultaneously in a single reaction vessel. Although emulsion PCR can also be used to amplify thousands of nucleic acids in a single reaction, emulsion PCR is limited by the impermeable nature of the oil surrounding aqueous droplets in the emulsion. Unlike an oil emulsion, the semipermeable membrane of the microcapsule of the present invention allows for the free exchange of low molecular weight molecules and reagents (e.g., dNTPs, fluorescently labeled ddNTPs, short primers) and reaction byproducts (e.g., pyrophosphate). As a result, the microcapsule of the present invention provides a near constant concentration of reaction reagents and substrates with the ability to remove inhibitory byproducts by diffusion. This property when coupled with Sanger sequencing biochemistry allows for the amplification and subsequent sequencing of longer nucleic acid templates than is possible with current emulsion PCR methods. For instance, the microcapsules of the present invention can be used to produce reads of about 800 to 1,000 high quality bases, whereas the 454 Life Sciences sequencing technique which uses emulsion PCR in conjunction with pyrosequencing is reported to only produce reads of about 100 to 200 high quality bases (Wicker, T. et al., 2006, BMC Genomics. 7: 275).
Microcapsules of the present invention each comprise a semipermeable membrane containing one or more enzymes and a nucleic acid template. The enzymes and nucleic acid template are located within an aqueous core of the microcapsule. The semipermeable membrane of the microcapsule allows for the free exchange of low molecular weight molecules (e.g., dNTPs, fluorescently labeled ddNTPs, short primers) and reaction byproducts (e.g., pyrophosphate). The semipermeable membrane, however, prevents enzymes and nucleic acid template from exiting the aqueous core of the microcapsule.
The present invention also includes methods for using microcapsules. For instance, the present invention includes methods of amplifying and sequencing a nucleic acid using a microcapsule as described herein.
Microcapsules of the invention are useful for enzyme-mediated reactions such as a polymerase-mediated reaction. For instance, the invention includes a thermostable microcapsule, which comprises a semipermeable membrane, an aqueous core, one or more polymerases in the aqueous core, and a nucleic acid template in the aqueous core. Microcapsules may be any appropriate shape or size, but a preferred microcapsule is spherical and approximately 1-10 μm in diameter. In one embodiment of the invention, the microcapsules are transparent.
Microcapsules are thermostable and capable of withstanding thermocycling for PCR. By “thermostable”, it is meant that the microcapsule can withstand high temperatures such as those required to denature nucleic acids. In another embodiment of the invention, the microcapsules are capable of withstanding high-speed flow sorting, for instance, sorting at greater than about 70,000/second. A collection of microcapsules of the invention are of relatively uniform size, i.e., are monodisperse, and have a diameter with a coefficient of variation of less than or about 10%.
Ideally, the semipermeable membrane of the microcapsule according to the invention is impermeable to high molecular weight molecules. On the other hand, the semipermeable membrane is permeable to low molecular weight molecules such as low molecular weight reagents. For instance, the semipermeable membrane can be permeable to molecules possessing a molecular weight of less than or about 20,000 g/mol, less than or about 10,000 g/mol, less than or about 5,000 g/mol, or less than or about 3,000 g/mol. In one embodiment of the invention, the semipermeable membrane is impermeable to molecules with a molecular weight of about 3,000 g/mol or more and is permeable to molecules with a molecular weight molecule of less than about 3,000 g/mol.
Alternatively, the semipermeable membrane of the present invention is impermeable to enzymes and nucleic acids that are longer than about 70 nucleotides in length. For instance, the semipermeable membrane is impermeable to the nucleic acid template contained within the aqueous core of the microcapsule.
The semipermeable membrane of the microcapsule is permeable to small molecular weight reagents and reaction byproducts. Thus, in sequencing, the semipermeable membrane is permeable to deoxynucleotide triphosphates (dNTPs), dideoxynucleotide triphosphates (ddNTPs), labeled ddNTPs, labels and dyes, pyrophosphates, divalent cations (e.g., magnesium ions and manganese ions), monovalent cations (e.g., potassium ions), and nucleic acids that are shorter than about 70 nucleotides in length.
The semipermeable membrane can comprise any polymer known in the art that is permeable to low molecular weight reagents and impermeable to high molecular weight reagents. It is important that the polymer not prevent an enzymatic reaction from occurring in the aqueous core of the microcapsule. Polymers that can be used, include, but are not limited to, acrylic polymers including, but not limited to crosslinked polyacrylamide, cyanoacrylate, diacrylates including poly(ethylene glycol) diacrylate (PEG-DA) and poly(ethylene glycol) dimethylacrylate (PEG-DMA) of various chain lengths. The semipermeable membrane can also comprise, for instance, epoxy resins including DuPont's “Somos 6100” series of resins. Porogens, such as various chain length poly(ethylene glycol)s can also be included to adjust the molecular weight cut off (MWCO) of the polymer shell of the microcapsules.
The semipermeable membrane can comprise a polymer that is capable of cross-linking to control the stability and MWCO of the microcapsule. In one embodiment of the invention, the polymer is a photocrosslinkable polymer.
The nucleic acid template within the aqueous core of each microcapsule serves as a substrate for the enzyme-mediated reaction. In one embodiment of the invention, there is a single nucleic acid template, i.e., one molecule, in each microcapsule. For instance, in one embodiment of the invention, the microcapsule comprises a semipermeable membrane, an aqueous core, one or more polymerase enzymes in the aqueous core, and one nucleic acid template in the aqueous core. In another embodiment of the invention, the microcapsule contains multiple copies of a single nucleic acid template.
The nucleic acid template can be either a DNA template or an RNA template, including genomic DNA, cDNA, mRNA, rRNA, tRNA, gRNA, siRNA, micro RNA, and others known in the art.
As can be appreciated by a skilled artisan, nucleic acid template can be a eukaryotic, prokaryotic, or viral nucleic acid; and may be a recombinant nucleic acid or a previously amplified nucleic acid fragment (e.g., PCR product).
Although the nucleic acid template may be derived from a clone, it is unnecessary to clone the nucleic acid molecule in vivo prior to use in the microcapsule of the present invention. For instance, sheared or enzymatically digested genomic nucleic acids may be used as nucleic acid templates.
The nucleic acid template can vary in length so long as it is of sufficient size to prevent it from crossing the semipermeable membrane. For instance, the nucleic acid template can be about or greater than 100 nucleotides in length, about or greater than 200 nucleotides in length, about or greater than 300 nucleotides in length, about or greater than 400 nucleotides in length, about or greater than 500 nucleotides in length, about or greater than 600 nucleotides in length, about or greater than 700 nucleotides in length, about or greater than 800 nucleotides in length, about or greater than 900 nucleotides in length, about or greater than 1000 nucleotides in length, about or greater than 1100 nucleotides in length, or about or greater than 1200 nucleotides in length. The invention includes microcapsules comprising a nucleic acid template that is at least about 100 nucleotides in length, at least about 200 nucleotides in length, at least about 300 nucleotides in length, at least about 400 nucleotides in length, at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, at least about 1000 nucleotides in length, at least about 2000 nucleotides in length, at least about 3000 nucleotides in length, at least about 4000 nucleotides in length, or at least about 5000 nucleotides in length.
The nucleic acid template may be either linear or circular, the circular topology having the added benefit of a reduced tendency to penetrate the polymer shell of the microcapsule.
The nucleic acid template should also contain at least one priming site for hybridization of a complementary primer oligonucleotide for DNA amplification.
The nucleic acid template may be single-stranded or double-stranded although the preferred template is single-stranded.
Microcapsules comprise one or more enzymes in the aqueous core. Examples of enzymes include nucleic acid modifying enzymes such as polymerases, reverse transcriptases, ligases, Klenow fragment and restriction endonucleases. Examples also include thermophilic DNA polymerases, such as Taq polymerase DNA polymerase I, DNA polymerase II, DNA polymerase III holenzyme, DNA polymerase IV, terminal transferase, Klenow fragment, T4 DNA polymerase, T7 DNA polymerase, BST DNA polymerase, and phi29 DNA polymerase. Additional examples of enzymes include various forms of “hot start” polymerases that are inactive at low temperatures (e.g., 40° C.) and only become active upon heating to relatively high temperatures (e.g., >90° C.).
In another embodiment of the invention, the enzymes are selected from a group of RNA polymerases, including, but not limited to, RNA polymerase I, RNA polymerase II, RNA polymerase III, and T7 RNA polymerase.
Microcapsules may contain more than one enzyme within the aqueous core. For instance, the present invention includes microcapsules with one, two, three, or four or more different enzymes within the aqueous core.
Microcapsules are permeable to low molecular weight reagents and buffers. Microcapsules further comprise one or more low molecular weight reagents. In one embodiment of the invention, the aqueous core is a buffer solution. Microcapsules can be stored or incubated in a solution comprising low molecular weight reagents and/or a buffer solution. Examples of low molecular weight reagents are described throughout this application and include dNTPs, ddNTPs, labeled ddNTPs (e.g., fluorescently labeled ddNTPs), divalent cations, monovalent cations, stabilizers and nucleic acid primers. Buffers that can be used with the microcapsules of the invention include polymerase buffers such as standard Taq buffer.
Microcapsules of the invention may be used with primers of any size. In other words, the microcapsules can be used with primers that are capable of passing through the membrane of the microcapsule as well as those are incapable of passing through the membrane of the microcapsule.
In one embodiment of the invention, the primers are able to pass through the semipermeable membrane of the microcapsule. Such primers can be up to about 70 nucleotides in length. For instance, primers that are about 5 to 10 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 10 to 30 nucleotides in length, 15 to 25 nucleotides in length, or 25 to 30 nucleotides in length can be used with the microcapsule of the invention as well as primers that are about 40 or fewer nucleotides in length, about 50 or fewer nucleotides in length, about 60 or fewer nucleotides in length, and about 70 or fewer nucleotides in length. In one embodiment of the invention, the primers are about 20 to 50 nucleotides in length.
In another embodiment of the invention, each microcapsule contains one or more primers that are unable to diffuse out of the aqueous core due to size. As can be appreciated by a skilled artisan, the size of the primers can vary depending on the polymer used as a semipermeable membrane. However, generally, primers greater than about 70 nucleotides are unable to cross the semipermeable membrane of the microcapsule.
The primers are substantially complementary or perfectly complementary to a region of the nucleic acid template. In one embodiment, the primer contains a small number of mismatches compared to the nucleic acid template that do not interfere with the ability of the primer to anneal to the nucleic acid template under stringent conditions. Such a primer may contain 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 mismatches compared to the nucleic acid template.
In one embodiment, universal primers can be used to hybridize with a common motif in the template. For example, the primer can be a poly-T primer that is capable of binding to a poly-A region in a template nucleic acid. Random primers are, of course, also useful.
The amplification reaction can be a PCR reaction (including rtPCR and others as described below) or can be another type of amplification, such as rolling circle amplification (RCA). Rolling circle amplification is described in U.S. Pat. Nos. 6,576,448; 6,977,153; and 6,287,824, each of which is incorporated by reference herein.
Microcapsules of the invention can be made by methods known in the art for forming membrane enclosed microcapsules, including, but not limited to, methods currently used for the microencapsulation of drugs.
In one embodiment of the invention, microcapsules are formed using a multiple sheath flow device as disclosed in co-owned U.S. patent application Ser. No. 12/221,792, filed on Aug. 6, 2008. U.S. patent application Ser. No. 12/221,792 discloses a multiple sheath flow device as illustrated in
In one embodiment of the invention, the microcapsules of the invention are formed using the flow focusing technology developed by Flow Focusing Incorporated (Menlo Park, Calif.) and Ingeniatrics S.L. (Sevilla, Spain). See, for instance, U.S. Pat. Nos. 6,557,834; 6,554,202; 6,450,189; 6,432,148; 6,405,936; 6,394,429; 6,386,463; 6,357,670; 6,299,145; 6,248,378; 6,241,159; 6,234,402; 6,197,835; 6,196,525; 6,189,803; 6,187,214; 6,174,469; 6,119,953; 6,116,516 and 6,464,886, each of which is herein incorporated by reference in its entirety.
Nucleic acid templates can be amplified in microcapsules of the invention. As can be appreciated by a skilled artisan, various nucleic acid amplification methods known in the art that employ an enzyme-mediated reaction can be easily modified for use with the present invention. The only requirement is that the one or more enzymes, nucleic acid template to be amplified, and, optionally, primers, be encapsulated within the semipermeable membrane of the microcapsule. For instance, in addition to polymerase chain reaction (PCR), other known amplifications methods such as rolling circle amplification (RCA), ligase chain reaction (European application EP 320 308), gap filling ligase chain reaction (U.S. Pat. No. 5,427,930), strand displacement amplification (U.S. Pat. No. 5,744,311) and repair chain reaction amplification (WO 90/01069) may be performed using the microcapsules of the present invention.
In one embodiment, microcapsules are used to amplify a nucleic acid template by polymerase chain reaction. In this case, the microcapsule contains a thermostable DNA polymerase (e.g., Taq polymerase) and a nucleic acid template for amplification. Preferably, the aqueous core and liquid surrounding the microcapsule contain a PCR buffer solution and dNTPs. Primers that are complementary to the nucleic acid template may be located within the aqueous core or, if capable of transversing the semipermeable membrane, in the PCR buffer solution bathing the microcapsule.
To perform PCR, one to over a billion microcapsules are placed in a tube with the appropriate PCR reagents. As with traditional PCR, the tube is placed in a thermocycler under conditions necessary for PCR (i.e., cycles of denaturation, annealing, and elongation). Briefly, in a thermocycler, the microcapsules are denatured by heating (e.g., 94° C. to 98° C.) for about 20 to 30 seconds. The microcapsules are then subjected to an annealing temperature (e.g., about 50° C. to 65° C.) for about 20 to 40 seconds. Elongation proceeds next. The elongation temperature (usually about 72° C. to 80° C.) and time (about 1,000 bases/minute) required for the elongation step depend on the polymerase enzyme used and length of nucleic acid template, respectively. The denaturation, annealing, and elongation steps are repeated several times (usually about 20 to 30 cycles) and may be capped off with an extended elongation step.
One or multiple nucleic acid templates may be amplified in a single reaction using one or a plurality of microcapsules. In one embodiment of the invention, each microcapsule contains multiple nucleic acid templates that are amplified by PCR (i.e., multiplex PCR). In a preferred embodiment of the invention, each microcapsule contains a single nucleic acid template that is amplified by PCR. In another preferred embodiment, billions of microcapsules, each microcapsule containing a single nucleic acid template, are amplified by PCR.
In a preferred embodiment, microcapsules are used to amplify a nucleic acid template by rolling circle amplification (RCA). In this case, the microcapsule contains a strand displacement DNA polymerase (e.g., phi29 polymerase) and a circular nucleic acid template for amplification. Preferably, the aqueous core and liquid surrounding the microcapsule contain a RCA buffer solution and dNTPs. A primer that is complementary to the circular nucleic acid template may be located within the aqueous core or, if capable of transversing the semipermeable membrane, in the RCA buffer solution bathing the microcapsule.
To perform RCA, one to over a billion microcapsules are placed in a tube with the appropriate RCA reagents. Isothermal amplification (e.g., 40° C.) of the circular template results in a linear concatamer of very high molecular weight that does not cross the polymer membrane of the capsule.
The microcapsules of the present invention can also be used in reverse transcriptase amplification reactions. In this embodiment of the invention, each microcapsule comprises a semipermeable membrane, an aqueous core, a reverse transcriptase and an RNA template for amplification.
In one embodiment of the invention, a starting nucleic acid template is amplified within a microcapsule as described above prior to sequencing. Although not necessary, one or more microcapsules that have previously been subjected to an amplification reaction can be “cleaned-up” prior to sequencing by dialyzing against a suitable buffer.
In one embodiment, microcapsules that have previously undergone amplification are used in a Sanger sequencing reaction. Depending on the number of templates to be sequenced, one to thousands, even millions or billions, of microcapsules are placed in a tube with sequencing reagents. Sequencing reagents include dNTPs, ddNTPs and primers. Preferably, the ddNTPs are fluorescently labeled so that all four ddNTPs can be incorporated in the growing DNA chain in a single reaction.
Depending on the desired read length of the sequencing reaction (e.g., 1,000 bases) and the sensitivity requirements of the fluorescence detection system (e.g., 1,000 labeled fragments per band), then the total number of Sanger extension products can be estimated (e.g., 1,000×1,000=1 million). If the initial single molecule template in each capsule has already been amplified to an equivalent number of copies (e.g., 1 million), then only a single cycle of polymerase extension and ddNTP termination will be required to produce the required number of Sanger extension products. If, however, the initial single molecule template in each microcapsule has been amplified to a lesser extent, then multiple cycles of polymerase extension and ddNTP termination can be employed using cycle sequencing to generate the necessary number of extension products. Cycle sequencing is performed in a thermocycler by methods known in the art.
Upon completion of the sequencing reaction, it is preferable that unincorporated labeled ddNTPs, dNTPs, primers and pyrophosphates are removed. In one embodiment of the invention, unwanted reagents and byproducts are removed by dialysis against a suitable buffer.
In one embodiment of the invention, it is preferred that each microcapsule contains a single starting nucleic acid template. Poisson statistics dictate the dilution requirements needed to insure that each microcapsule contains only a single starting nucleic acid template. For example, if, on average, each microcapsule is to contain only a single template, about ⅓ of the microcapsules will be empty and contain no nucleic acid template, about ⅓ will contain exactly one nucleic acid template, and about ⅓ will contain two or more templates.
The microcapsule population may be enriched to maximize the fraction that started with a single nucleic acid template. Because the Sanger sequencing reaction incorporates fluorescently labeled ddNTPs, it is possible to flow sort the microcapsules after sequencing (and, preferably, after a purification step) to enrich for those that are fluorescent rather than empty. High-speed flow sorters, such as the MoFlo (Beckman-Coulter, Inc., Fullerton, Calif.), are capable of sorting at rates in excess of 70,000 per second and can be used to enrich a population of microcapsules of the invention. Similarly, it is possible to exploit other differences between empty and full microcapsules (e.g., buoyant density) to enrich a population of microcapsules. In order to enrich for microcapsules with one starting nucleic acid template as opposed to several different starting templates, it may be desirable to skew the Poisson distribution accordingly.
Electrophoresis-based sequencers and non-electrophoresis-based sequencers can be used to determine the sequence of the nucleic acid product contained within each of the microcapsules. In one embodiment of the invention, microcapsules are loaded either manually or automatically into the sequencer and broken in situ to release their contents. In another embodiment of the invention, microcapsules are loaded manually or automatically into the sequencer without the need for breaking the microcapsules. Instead, the application of an electric field during electrophoresis causes the Sanger extension products to reptate end-on through the pores in the semi-permeable membrane, even though they would not normally diffuse through such pores without the application of such an externally applied field.
Electrophoresis methods, including slab gel-based, capillary-based and microchip-based methods known in the art, can be used to determine the sequence of one or more microcapsules. Capillary-based methods include commonly used sequencers such as the Applied Biosystems 3730 Series DNA analyzers as well as newer massively parallel continuous electrophoresis systems.
The microcapsules described herein can be used to perform thousands, even millions or billions, of Sanger sequencing reactions in a single tube. In one preferred embodiment of the invention, a massively parallel electrophoresis system is used to read each sequence. One such method is continuous film electrophoresis, which is described in co-owned U.S. patent application Ser. No. 12/221,794, filed Aug. 6, 2008. In one embodiment of the invention, microcapsules, or the sequenced product from microcapsules, are loaded on to a continuous film electrophoresis sequencer for sequence determination.
In yet another embodiment of the invention, the sequence of the nucleic acid of one or more microcapsules is determined in real time (i.e., sequencing-by-synthesis). In this embodiment, the sequencing reaction and determination of the sequence occur simultaneously.
In a further embodiment, the present invention provides kits containing microcapsules. In one embodiment, the kit comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition, which includes a microcapsule or plurality of microcapsules as described herein.
The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial end user standpoint, including, but not limited to, buffers, reagents, and package inserts with instructions for use. In one embodiment of the invention, the kit contains dNTPs and/or ddNTPs.
The following example demonstrates an embodiment of the manufacture and use of microcapsules according to the invention.
Three model NE500 syringe pumps (New Era Pump Systems, Inc., Wantagh, N.Y.) controlled by a PC running WinPumpControl software (Open Cage Software, Inc., Huntington, N.Y.) deliver fluids to the flow focusing nozzle inlet fittings illustrated in
To form impermeable polymer shell microcapsules, the following three solutions were delivered to the flow focusing nozzle at the indicated volumetric flow rates: (1) Core Solution—sodium fluorescein (5 mg/mL—Fluka/Sigma-Aldrich, St. Louis, Mo.), glycerin (25% v/v—Walgreens, Deerfield, Ill.) in distilled water at 0.1 mL min−1; (2) Polymer Shell Solution—PEGDMA 200 (polyethylene glycol 200 dimethacrylate) (Monomer-Polymer & Dajak Labs, Inc., Feasterville, Pa.), 4.76% v/v 2-hydroxy-2-methyl propiophenone (Sigma-Aldrich, St. Louis, Mo.), 0.4% v/v TEMED (N,N,N′,N′-tetramethylethylenediamine) and 0.05 g/mL 2,2-dimethoxy-2-phenyl-acetophenone (Sigma-Aldrich, St. Louis, Mo.) at 0.15 ml min−1; and (3) Focusing Fluid—1% poly(vinyl alcohol) 87-89% hydrolyzed (Typical Mw 85,000-124,000) (Sigma-Aldrich, St. Louis, Mo.) in distilled water at 6.5 mL min−1. The orifice of the flow focusing nozzle is positioned ˜15 cm above the liquid surface of a 100 mL beaker containing 50 mL of the Focusing Fluid to collect the microcapsules. The beaker sits on a near-UV transilluminator (Spectroline Slimline™ Series 365-8W, Spectronics Corporation, Westbury, N.Y.) to provide 360 nm illumination for photoinitiation. In addition, two 40W “black light” fluorescent lamps (GE F40BLB—General Electric Company, Fairfield, Conn.) are positioned ˜10 cm to the side of the emerging liquid jet to photoinitiate polymerization of the shell of the microcapsule prior to “splash down” in the collection beaker. Aliquots (35 μL) of microcapsules are mounted on a microscope slide and examined in an Axiovert 25 inverted microscope (Carl Zeiss Microlmaging, Inc., Thornwood, N.Y.). Digital images are captured using an AxioCam CCD camera (Carl Zeiss Microlmaging, Inc., Thornwood, N.Y.) and analyzed using AxioVision software Ver 4.6 (Carl Zeiss Microlmaging, Inc., Thornwood, N.Y.). Fluorescence imaging employs a 50W Hg lamp illuminator (Carl Zeiss Microlmaging, Inc., Thornwood, N.Y.) and a FITC filter cube (484 nm excitation, 494/521 nm emission) (Carl Zeiss Microlmaging, Inc., Thornwood, N.Y.). Example bright field and fluorescence images of impermeable polymer shell microcapsules generated using the above described system are provided in
Smaller diameter impermeable polymer shell microcapsules are generated by adjusting the relative flow rates of the solutions from Example 1 as follows: Core Solution—0.025 ml min−1; Polymer Shell Solution—0.05 ml min−1; and Focusing Fluid—6.0 ml min−1. The resulting microcapsules, illustrated in
Intermediate diameter and/or thinner shell impermeable polymer microcapsules can also be produced using an alternative Polymer Shell Solution, blending PEGDMA 200 with PEGDA (poly(ethylene glycol) diacrylate of different chain lengths (PEGDA575−Mn˜575 or PEGDA700−Mn˜700—Sigma-Aldrich, St. Louis, Mo.) in the ratio of 4:1 PEGDMA 200:PEGDAXXX and by adjusting the relative flow rates of the three solutions The Core Solution is composed of a low molecular weight fluorescent marker (sodium fluorescein Mw=376 Da) and a high molecular weight marker (rhodamine B isothiocyanate-labeled dextran Mw=10 kDa) loaded together into the microcapsules in approximately equimolar amounts using the following composition: sodium fluorescein (0.26 mg/mL—Fluka/Sigma-Aldrich, St. Louis, Mo.) and rhodamine B isothiocyanate-dextran (5 mg/mL—Sigma-Aldrich, St. Louis, Mo.) and glycerol (25% v/v—Sigma, St. Louis, Mo.) in distilled water. Intermediate size microcapsules measuring ˜50 μm in diameter were produced using PEDGA575 with the following flow rates: Core Solution—0.05 ml min−1, Polymer Shell Solution—0.010 ml min−1, and Focusing Fluid—15 ml min−1. The resulting microcapsules are shown in
Permeable microcapsules are generated under identical conditions to Example 1 except for the addition of 5% v/v acrylic acid (Sigma-Aldrich, St. Louis, Mo.) to the Polymer Shell Solution as shown in
Semi-permeability of the polymer shell of the microcapsules as produced in Example 4 was demonstrated by comparing the relative loss/retention of a low molecular weight fluorescent marker (sodium fluorescein Mw=376 Da) and a high molecular weight marker (rhodamine B isothiocyanate-labeled dextran Mw=10 kDa) loaded together into the semi-permeable microcapsules in approximately equimolar amounts as described in Example 3. All conditions were identical to those in Example 4, except for the composition of the Core Solution, which was modified as follows: sodium fluorescein (0.26 mg/mL—Fluka/Sigma-Aldrich, St. Louis, Mo.) and rhodamine B isothiocyanate-dextran (5 mg/mL—Sigma-Aldrich, St. Louis, Mo.) and glycerol (25% v/v—Sigma, St. Louis, Mo.) in distilled water. Impermeable micorcapsules were prepared as controls using conditions identical to those provided in Example 1 with the Core Solution detailed above.
The harvested microcapsules were imaged directly in Focusing Fluid without washing ˜5 minutes after they were created. The microcapsules were then stored at room temperature in Focusing Fluid for ˜16 hours and reimaged. Brightfield and fluorescence images are provided in
There was substantial loss of fluorescein within 5 minutes from the semi-permeable capsules compared with the impermeable control microcapsules, while there was no obvious loss of the rhodamine-labeled dextran even after 16 hours in the semi-permeable microcapsules, indicating that these microcapsules were preferentially permeable to the lower molecular weight fluorescein while retaining the higher molecular weight rhodamine-labeled dextran polymer. The Molecular Weight Cut Off (MWCO) of the semipermeable polymer shell membrane of these microcapsules is therefore >400 Daltons but <10,000 Daltons.
Altered permeability characteristics of polymer shell microcapsules were demonstrated as described in Example 2 using an alternative Polymer Shell formulation. All conditions were identical, except for the composition of the Core Solution, which was modified as follows: fluorescein isothiocyanate-dextran (2 mg/mL—Fluka/Sigma-Aldrich, St. Louis, Mo.) and rhodamine B isothiocyanate-dextran (5 mg/mL—Sigma-Aldrich, St. Louis, Mo.) and glycerol (25% v/v—Sigma, St. Louis, Mo.) in distilled water, and the Polymer Shell Solution, which was modified as follows: 2:1 v/v PEGDMA 200 and MPEOEA (methoxypoly(ethyleneoxy) ethyl acrylate) (Monomer-Polymer & Dajak Labs, Inc., Feasterville, Pa.).
The harvested microcapsules were imaged directly in Focusing Fluid without washing ˜5 minutes after they were created. The microcapsules were then stored in the dark at room temperature in Focusing Fluid for ˜24 hours and reimaged. Brightfield and fluorescence images are provided in
There was no significant loss of either fluorescent signal from the permeable capsules within 5 minutes compared with the impermeable control microcapsules. However, at t=24 hrs, both signals had decreased significantly with this polymer shell formulation. The Molecular Weight Cut Off (MWCO) of the permeable polymer shell membrane of these microcapsules is therefore >10,000 Daltons.
Semi-permeability of the polymer shells of the microcapsules was further demonstrated by encapsulation of high molecular weight DNA (single-stranded M13mp18 DNA—Mw 2.4 MDa, 7,249 bases) in the microcapsules and then labeling the DNA inside the microcapsules by incubating them in an exogenously added, low molecular weight fluorescent dye specific for single-stranded DNA (OliGreen®, Mw<1,000 Da—Invitrogen/Molecular Probes, Eugene, Oreg.). Semi-permeable microcapsules were produced as described in Example 5. All conditions were identical, except for the composition of the Core Solution, which was modified as follows: single-stranded M13mp18 DNA (20 μg/mL—Sigma-Aldrich, St. Louis, Mo.) in 1×TE buffer (10 mM Tris (TRIZMA®—tris(hydroxymethyl)aminomethane hydrochloride—Sigma-Aldrich, St. Louis, Mo.), 1 mM EDTA (ethylenediamenetetraacidic acid—Sigma-Aldrich, St. Louis, Mo.), pH 8.1) containing glycerol (25% v/v—Sigma-Aldrich, St. Louis, Mo.), and the Polymer Shell Solution, which was modified as follows: 10:1 v/v PEGDMA 200 and MPEOEA (methoxypoly(ethyleneoxy) ethyl acrylate) (Monomer-Polymer & Dajak Labs, Inc., Feasterville, Pa.). Negative control microcapsules were made with a Core Solutions containing only TE buffer and glycerin.
The harvested microcapsules were decanted and rinsed 2× with 20 mL distilled water. Negative control microcapsules without DNA and microcapsules containing the DNA Core Solution were incubated by mixing 100 μL of microcapsule suspension with 40 μL of a 1:20 dilution of OliGreen® in TE buffer. Fluorescence images were taken after 1 hour of incubation in the dark at room temperature, and are provided in
There was no observable fluorescence from OliGreen® when added exogenously to negative control microcapsules without DNA. Microcapsules containing 20 μg/mL of single-stranded M13 DNA were brightly stained after incubation for 1 hour in exogenously added OliGreen®, indicating that these microcapsules were preferentially permeable to the lower molecular weight OliGreen® dye while retaining the much higher molecular weight single-stranded M13 DNA. The Molecular Weight Cut Off (MWCO) of the semi-permeable polymer shell membrane of these microcapsules is therefore >1,000 Daltons but <2.4 million Daltons.
DNA amplification in semi-permeable polymer shell microcapsules was demonstrated using hyperbranched Rolling Circle Amplification (RCA). High molecular weight DNA (single-stranded M13mp18 DNA—Mw 2.4 MDa, 7,249 bases) was incorporated in microcapsules along with φ29 polymerase, random hexamers as primers, and deoxynucleotide triphosphate mix (dNTP mix). Semi-permeable microcapsules were produced as described in Example 4. All conditions were identical, except for the composition of the Core Solution, which was modified as follows: RCA Mix formulated by combining 13.5 μL diluted single-stranded M13mp18 DNA (1 μg/mL—Sigma-Aldrich, St. Louis, Mo.), 2.125 μL concentrated φ29 polymerase in buffer (New England Biolabs, Ipswich, Mass.), 8.75 μL random hexamer primers in H2O (New England Biolabs, Ipswich, Mass.), 8.75 μL glycerol (25% v/v—Sigma-Aldrich, St. Louis, Mo.), 3.75 μL 10×RCA buffer (37 mM TRIS-HCl, 50 mM KCl, 10 mM MgCl2, 5 mM NH2SO4, 1 mM DTT (dithiothrietol), 1×BSA), 0.4 μL 10×BSA (bovine serum albumin—New England Biolabs, Ipswich, Mass.) and 3.75 μL dNTP mix (New England Biolabs, Ipswich, Mass.), and the Focusing Fluid, which was composed of 5 wt % PVA in 1×RCA buffer. DNA cannot be visually detected at this low initial concentration in polymer microcapsules.
The harvested microcapsules were split into four 100 μL batches in 500 μL Safe-Lock Eppendorf microfuge tubes (Brinkmann Instruments, Inc., Westbury, N.Y.). The first batch was incubated as described below with no further treatment. This polymer microcapsule formulation is known to be permeable to dye molecules that are approximately the same molecular weight as native nucleotides. Therefore, 25 μl dNTP mix was added externally to the second and fourth batches. The third and fourth batches were then subjected to a five minute heat inactivation of the φ29 polymerase at 65° C. The four microfuge tubes containing the microcapsules were incubated at 30° C. for 4 hours in a thermocycler (MiniCycler™—MJ Research, Watertown, Mass.). Following incubation, 100 μL of 2× OliGreen® reagent (Invitrogen/Molecular Probes, Eugene, Oreg.) in 1×TE was added to each tube, incubated for ˜16 hours at room temperature and then imaged with 4 second exposures. Fluorescence images are provided in
There was no observable fluorescence from OliGreen® when added exogenously to heat-inactivated control microcapsules, either without exogenously added dNTPs (
Hyperbranched Rolling Circle Amplification (RCA) was further demonstrated with an alternative polymer shell formulation. Conditions were identical to those in Example 8 except for the composition of the Polymer Shell solution, which was identical to that used in Example 6, except for the ratio of PEGDMA 200 and MPEOEA, which was 10:1 v/v. The microfuge tubes containing the microcapsules were incubated at 35° C. for 10 hours in a thermocycler (MiniCycler™—MJ Research, Watertown, Mass.). Following incubation, 60 μL of 1:20 dilution of OliGreen® reagent (Invitrogen/Molecular Probes, Eugene, Oreg.) in 1×TE was added to each tube, incubated for ˜3 hours at room temperature and then imaged (exposure time=3 sec). Brightfield and fluorescence images are shown in
Thermostability of semi-permeable polymer shell microcapsules was demonstrated by producing FITC-labeled dextran (4 kDa) loaded microcapsules as described in Example 5. The harvested microcapsules were rinsed in distilled water and imaged ˜5 minutes after they were created. The microcapsules were then heated to ˜95° C. in distilled water for 20 minutes, cooled to room temperature and reimaged. Brightfield and fluorescence images are provided in
There was no observable loss of fluorescence or change in the morphology of the microcapsules after heating, indicating that they are sufficiently thermostable to withstand conditions for PCR and/or cycle sequencing.
Permeability of the alternative formulation polymer shell microcapsules to dye-labeled dideoxynucleotide terminators was demonstrated using conditions identical to those in Example 7 except for the composition of the Core Solution, which was 25% v/v glycerol. Impermeable microcapsules were used as controls.
5 μL of suspended microcapsules were mixed with 5 μL of dye-labeled dideoxynucleotide terminators (tetramethyl rhodamine-ddTTP, Thermo Sequenase Dye Terminator Cycle Sequencing Core Kit, Amersham Biosciences, Piscataway, N.J.) and incubated at room temperature in the dark for 3 hours.
Microcapsules were then washed with 2 mL distilled H2O and imaged immediately. Microcapsules were allowed to incubate in distilled H2O for an additional 20 hours in the dark and imaged again as shown in
The aqueous cores of the impermeable PEGDMA microcapsules were non-fluorescent after 3-hour incubation in dye-labeled ddTTP, whereas the aqueous cores of the semi-permeable PEGDMA-MPEOEA microcapsules show significant internal fluorescence. The process is reversible, as indicated by the loss of internal fluorescence upon further incubation in water, indicating that dye-labeled dideoxynucleotide terminators can freely exchange across the semi-permeable polymer shell membrane of these microcapsules.
Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, patent applications and publications referred to in this application are herein incorporated by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12/221791 | Aug 2008 | US | national |
This application is related to and claims the benefit of U.S. nonprovisional patent application Ser. No. 12/221,791, filed Aug. 6, 2008, the contents of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/52936 | 8/6/2009 | WO | 00 | 4/28/2011 |
