SYSTEM FOR BREAKING STABILIZED EMULSIONS
`
`Cross-References
`
`This application incorporates by reference in their entireties for all purposes the
`
`following materials: U.S. Patent No. 7,041,481,
`
`issued May 9, 2006; U.S. Patent
`
`Application Publication No. 2010/0173394 A1, published July 8, 2010; U.S. Patent
`
`Application Serial No. 12/976,827, filed December 22, 2010; and Joseph R. Lakowicz,
`
`PRINCIPLES OF FLUORESCENCE SPECTROSCOPY(2™ Ed. 1999).
`
`Introduction
`
`10
`
`Aqueous droplets can be suspendedin oil to create a water-in-oil emulsion. The
`
`emulsion can be stabilized with a surfactant and/or protein, among others, to reduce
`
`coalescenceof droplets during heating, cooling, and transport, thereby enabling thermal
`
`cycling to be performed. Accordingly, emulsions have been used to perform single-copy
`
`amplification of nucleic acid target molecules in droplets using the polymerase chain
`
`15
`
`reaction (PCR).
`
`Compartmentalization of single molecules of a nucleic acid target in droplets of
`
`an emulsion alleviates problems encountered in amplification of larger sample volumes.
`
`In particular, droplets can promote more efficient and uniform amplification of targets
`
`from samples containing complex heterogeneous nucleic acid populations, because
`
`20
`
`sample complexity in each droplet is reduced. The impact of factors that lead to biasing
`
`in bulk amplification, such as amplification efficiency, G+C content, and amplicon
`
`annealing, can be minimized by droplet compartmentalization. Unbiased amplification
`
`can be critical
`
`in detection of rare species, such as pathogens or cancer cells,
`
`the
`
`
`
`Cross-References
`
`This application incorporates by reference in their entireties for all purposes the
`
`following materials: U.S. Patent No. 7,041,481,
`
`issued May 9, 2006; U.S. Patent
`
`Application Publication No. 2010/0173394 A1, published July 8, 2010; U.S. Patent
`
`Application Serial No. 12/976,827, filed December 22, 2010; and Joseph R. Lakowicz,
`
`PRINCIPLES OF FLUORESCENCE SPECTROSCOPY(2™ Ed. 1999).
`
`Introduction
`
`10
`
`Aqueous droplets can be suspendedin oil to create a water-in-oil emulsion. The
`
`emulsion can be stabilized with a surfactant and/or protein, among others, to reduce
`
`coalescenceof droplets during heating, cooling, and transport, thereby enabling thermal
`
`cycling to be performed. Accordingly, emulsions have been used to perform single-copy
`
`amplification of nucleic acid target molecules in droplets using the polymerase chain
`
`15
`
`reaction (PCR).
`
`Compartmentalization of single molecules of a nucleic acid target in droplets of
`
`an emulsion alleviates problems encountered in amplification of larger sample volumes.
`
`In particular, droplets can promote more efficient and uniform amplification of targets
`
`from samples containing complex heterogeneous nucleic acid populations, because
`
`20
`
`sample complexity in each droplet is reduced. The impact of factors that lead to biasing
`
`in bulk amplification, such as amplification efficiency, G+C content, and amplicon
`
`annealing, can be minimized by droplet compartmentalization. Unbiased amplification
`
`can be critical
`
`in detection of rare species, such as pathogens or cancer cells,
`
`the
`
`
`
`presence of which could be masked by a high concentration of background species in
`
`complexclinical samples.
`
`A stabilized emulsion can withstand the repetitive cycles of heating and cooling
`
`that drive PCR amplification, without substantial loss of droplet integrity. However,
`
`it is
`
`often desirable to harvest nucleic acid from the emulsion after amplification for further
`
`analysis, such as by sequencing. In this case, the emulsion needs to be destabilized or
`
`“broken,” to transform the dispersed aqueous phase into a continuous aqueous phase
`
`in which the amplified nucleic acid is pooled. Emulsions that are stable enough for PCR
`
`amplification can be difficult to break.
`
`10
`Approaches are needed to isolate amplified nucleic acids from_stabilized
`
`emulsions for further reaction and/or analysis.
`
`Summary
`
`The present disclosure provides a system, including methods and apparatus, for
`
`breaking an emulsion by mixing the emulsion with an organic solvent followed by
`
`15
`
`centrifugation.
`
`
`
`DetailedDescription
`
`The present disclosure provides a system, including methods and apparatus, for
`
`breaking an emulsion by mixing the emulsion with an organic solvent followed by
`
`centrifugation. The emulsion may be generated in a stabilized form, such as in the
`
`20
`
`presence of surfactant and/or protein,
`
`to maintain the integrity of aqueous droplets
`
`during droplet manipulation and/or performance of a reaction (e€.g., nucleic amplification
`
`through thermal cycling). The organic solvent destabilizes the droplets of the emulsion,
`
`such as by removal of a proteinaceous skin that surrounds each droplet. As a result, the
`
`
`
`droplets fuse to form a continuous aqueous phase, which may be removed from the
`
`organic solvent and/or the carrier phase of the emulsion. Nucleic acid from the
`
`continuous aqueous phase then may be combined with one or more other reagents to
`
`perform another
`
`reaction(s)
`
`(e€.g.,
`
`sequencing), may be resolved into different
`
`components (e.g., by size), and/or may be analyzed with a detector.
`
`The procedure for emulsion breakage disclosed herein may have numerous
`
`advantages over other breakage approaches, such as increased speed, moreefficient
`
`recovery of droplet components, and/or reduced cost, among others.
`
`Further aspects of the present disclosure are presented in the following sections:
`
`10
`
`(1) overview of exemplary methods, and (Il) examples.
`
`I.
`
`Overview of Exemplary Methods
`
`This section provides an overview of exemplary methods of breaking emulsions
`
`containing amplified nucleic acids. The method steps disclosed in this section and
`
`elsewhere in the present disclosure may be performed in any suitable combination,
`
`in
`
`15
`
`any suitable order, and any number oftimes.
`
`An emulsion may be generated. The emulsion may include aqueous droplets
`
`disposed in an immiscible continuous phase (e.g., a fluorocarbon oil). Each aqueous
`
`droplet may provide a reaction chamber in which to perform a reaction, such as nucleic
`
`acid amplification. In other words, each droplet may be configured to amplify a nucleic
`
`20
`
`acid target,
`
`if present,
`
`in the droplet. The aqueous droplets may be stabilized against
`
`coalescence (fusion) by the presence of one or more surfactants and/or proteins in the
`
`emulsion.
`
`In some embodiments, each droplet may be encapsulated individually by a
`
`
`
`skin, which may be a proteinaceous skin, disposed at the interface between the droplet
`
`and the immiscible continuous phase.
`
`The emulsion may be subjected to reaction conditions that promote occurrence
`
`of a reaction in the droplets. For example, the emulsion (and/or droplets thereof) may be
`
`heated to, or incubated at, one or more elevated temperatures (i.e., above room
`
`temperature),
`
`to promote nucleic
`
`acid amplification in
`
`the droplets.
`
`In
`
`some
`
`embodiments,
`
`the emulsion may be thermally cycled to promote nucleic acid
`
`amplification by the polymerase chain reaction.
`
`The droplets may be detected. For example, signals may be generated from the
`
`10
`
`droplets based on light detected from the droplets. The signals may correspond to
`
`whether or not nucleic acid amplification occurred in particular droplets. Light may be
`
`detected from individual droplets, such as detected in-parallel with droplets disposed in
`
`a substantially two-dimensional arrangement, or detected serially with droplets disposed
`
`in a flow channel, among others.
`
`In some cases, the emulsion may be broken without
`
`15
`
`generating signals from the droplets.
`
`The emulsion may be contacted with an organic solvent (also termed an organic
`
`phase) including one or more organic compounds (including an organic liquid). The
`
`organic solvent may be selected according to the composition of the emulsion, for
`
`example, based on the particular oil present in the continuous phase and whether or not
`
`20
`
`the droplets are stabilized by a proteinaceous skin. The organic solvent may be
`
`immiscible/substantially insoluble (e.g., less than about 5, 2, or 1% soluble) or miscible
`
`with the aqueous phase of the droplets and miscible or immiscible with the continuous
`
`phase of the emulsion. The organic phase may be capable of denaturing proteins.
`
`In
`
`
`
`exemplary embodiments, the organic solvent may include or be a halogen-substituted
`
`hydrocarbon (e.g., chloroform, a fluorine-substituted hydrocarbon, another low-viscosity
`
`halocarbon, or the like), an aliphatic alcohol (e.g., n-butanol, isobutanol, isopropanol, or
`
`ethanol), an aromatic alcohol (e.g., phenol), an ether (e.g., diethyl ether), a ketone (e.g.,
`
`acetone), or an ester (e.g., ethyl acetate), among others.
`
`The emulsion may be mixed, such as mixed vigorously, with the organic solvent.
`
`Vigorous mixing may be effected by shaking, vortexing, sonicating, stirring, or the like.
`
`In some cases, at least one more aliquot of the organic solvent may be added to the
`
`emulsion and the step of mixing repeated.
`
`10
`
`The emulsion mixed with the organic solvent may be centrifuged to promote
`
`separation of phases. Centrifugation may achieve any suitable g-force (e.g., at least
`
`about 1, 2, 5, or 10 thousand times the force of gravity, among others) for any suitable
`
`time period (e.g., at least about 1, 2, 5, 10, 30, or GO seconds, among others). The
`
`separated phases may include a continuous aqueous phase and at least one other
`
`15
`
`phase (e.g.,
`
`the organic solvent and the immiscible continuous phase (or aqueous
`
`phase) combined as a single phase or as respective distinct phases). The continuous
`
`aqueous phase may be disposed above or below one or more other phases. For
`
`example, the aqueous phase may be positioned as the top phase,
`
`the intermediate
`
`phase, or the bottom phase. Precipitated protein may collect at an interface between the
`
`20
`
`aqueous phase and another liquid phase.
`
`The continuous aqueous phase may be isolated from the other phase(s). For
`
`example, at least a portion of the continuous aqueous phase (or the other phase(s))
`
`may be removed from a container holding the separate phases. Removal may be
`
`
`
`effected by a fluid transfer device, such as a pipet. The non-aqueous phase(s) may be
`
`extracted one or more times with additional aqueous fluid to recover more of the
`
`continuous aqueous phase and/or nucleic acid therein.
`
`The isolated aqueous phase may be treated to eliminate a small amount of
`
`organic solvent and/or or immiscible continuous phase that contaminates the aqueous
`
`phase. For example, the aqueous phase may be contacted with a chromatography
`
`matrix (e€.g.,
`
`a size-exclusion matrix, an ion-exchange matrix, or the like) to remove
`
`residual amounts of the unwanted compounds. Alternatively, or in addition, nucleic acid
`
`may be separated from the aqueous phase, such as by contact with a chromatography
`
`10
`
`matrix, precipitation of nucleic acid (e.g., with an alcohol), or the like.
`
`Nucleic acid obtained from the isolated aqueous phase may be processed and/or
`
`analyzed. For example, the nucleic acid may be sequenced, sized by chromatography
`
`(e.g., by gel electrophoresis), further amplified,
`
`ligated, or any combination thereof,
`
`among others.
`
`15
`
`Further aspects of generating stabilized emulsions, performing nucleic acid
`
`amplification in droplets, droplet detection, and processing droplet signals are described
`
`in the materials listed above under Cross-References, which are incorporated herein by
`
`reference, particularly U.S. Patent Application Publication No. 2010/0173394 A‘,
`
`published July 8, 2010; and U.S. Patent Application Serial No. 12/976,827,
`
`filed
`
`20
`
`December 22, 2010.
`
`Il.
`
`Examples
`
`This section presents selected aspects and embodiments of
`
`the present
`
`disclosure related to methods of breaking an emulsion with an organic solvent.
`
`
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