CN116064734A

CN116064734A - Nucleic acid amplification

Info

Publication number: CN116064734A
Application number: CN202310142485.7A
Authority: CN
Inventors: C-Y·李; D·拉夫; S-M·陈; J·奥尼尔; R·卡辛斯卡斯; J·罗恩伯格
Original assignee: Life Technologies Corp
Current assignee: Life Technologies Corp
Priority date: 2012-04-19
Filing date: 2013-03-15
Publication date: 2023-05-05
Also published as: CN104471075B; CN109486902B; SG10201802883UA; WO2013158313A1; CN114854832A; CN104471075A; SG11201406717RA; CN109486902A

Abstract

In some embodiments, the present teachings provide methods for nucleic acid amplification, including forming a reaction mixture, and subjecting the reaction mixture to conditions suitable for nucleic acid amplification. In some embodiments, a method for nucleic acid amplification comprises subjecting a nucleic acid to be amplified to partial denaturing conditions. In some embodiments, the method for nucleic acid amplification comprises performing the amplification without completely denaturing the amplified nucleic acid. In some embodiments, the methods for nucleic acid amplification use enzymes and polymerases that catalyze homologous recombination. In some embodiments, the method for nucleic acid amplification may be performed in a single reaction vessel. In some embodiments, the method for nucleic acid amplification may be performed in a single continuous liquid phase of the reaction mixture without the need for compartmentalization of the reaction mixture or immobilization of the reaction components. In some embodiments, the method for nucleic acid amplification comprises amplifying at least one polynucleotide on a surface under isothermal amplification conditions, optionally in the presence of a polymer. The polymer may include a sieving agent and/or a diffusion reducing agent.

Description

Nucleic acid amplification

Background

This application is a divisional application with application number 201380031868.1.

Nucleic acid amplification is very useful in molecular biology and has wide applicability in virtually every aspect of biology, therapeutics, diagnostics, forensics, and research. Typically, one or more primers are used to generate an amplicon from a starting template, wherein the amplicon corresponds to or is complementary to the template from which the amplicon was generated. Multiplex amplification also simplifies the process and reduces costs. The application relates to methods and reagents for nucleic acid amplification and/or analysis.

Summary of The Invention

Provided herein are methods, reagents, and products for nucleic acid amplification and/or analysis. Amplification may utilize immobilized and/or dissolved primers. A single set of primers may be mixed with different templates, or a single template may be contacted with multiple different primers, or multiple different templates may be contacted with multiple different primers. Amplicons produced from the methods provided herein are suitable substrates for further analysis, e.g., sequencing.

In some embodiments, the present teachings provide compositions, systems, methods, devices, and kits for nucleic acid amplification.

Detailed description of the drawings

Fig. 1 provides a schematic diagram showing an embodiment of template walking (template walking). In an alternative embodiment, the immobilized primer comprises a primer designated (A) _n Adenosine-rich sequences of (A), for example ₃₀ And the primer binding site on the template for the immobilized primer comprises a complementary T-rich sequence, e.g. (T) ₃₀ 。

FIG. 2 depicts a schematic of amplification by template walking on beads and stacking of beads on a planar array for sequencing.

Fig. 3 depicts some alternative embodiments of semiconductor-based detection using sequencing by synthesis. Template walking can be used to create a population of clonal amplicons on beads or on the base or bottom of a reaction chamber. In an alternative embodiment, the immobilized primer comprises a primer designated (A) _n Adenosine-rich sequences of (A), for example ₃₀ And the primer binding site on the template for the immobilized primer comprises a complementary T-rich sequence, e.g. (T) ₃₀ 。

FIG. 4 depicts some alternative embodiments of the immobilization site on a planar substrate in the form of a primer lawn. A single continuous lawn that may use an array of discrete fixation sites or primers may be considered a random array of fixation sites. Optionally, the location of one or more fixation sites in the continuous lawn of primers may be as yet undetermined, wherein the location is determined at the time of initiation of attachment of the template prior to walking, or by the space occupied by the amplified clusters. In an alternative embodiment, the immobilized primer comprises a primer designated (A) _n Adenosine-rich sequences of (A), for example ₃₀ And the primer binding site on the template for the immobilized primer comprises a complementary T-rich sequence, e.g. (T) ₃₀ 。

FIG. 5 shows the effect of temperature on the template walking reaction. Delta Ct before and after template walking amplification is calculated and plotted against reaction temperature.

FIG. 6 provides a table of Ct values for 96 duplex TaqMan qPCR reactions.

Figure 7 depicts data showing about 100,000-fold amplification by template walking on beads. Delta Ct before and after the template walking reaction and amplification fold before and after the template walking reaction were calculated and plotted for the reaction time.

FIG. 8 provides a schematic depiction of an exemplary chain flipping and walking strategy. (A) template walking, (B) strand inversion to generate inverted strands, (C) addition of a new primer binding sequence Pg' on the final inverted strand.

FIG. 9 depicts Ion Torrent from a polynucleotide template amplified using a recombinase-mediated amplification reaction ^TM Exemplary read length histogram for PGM sequencing runs.

FIG. 10 depicts Ion Torrent from a polynucleotide template amplified using a recombinase-mediated amplification reaction ^TM Exemplary read length histogram for the Proton sequencing run.

FIG. 11 depicts Ion Torrent from a polynucleotide template amplified using a recombinase-mediated amplification reaction ^TM Exemplary read length histogram for the Proton sequencing run.

FIG. 12 depicts Ion Torrent from a polynucleotide template amplified using a recombinase-mediated amplification reaction ^TM Exemplary read length histogram for the Proton sequencing run.

Fig. 13 includes an illustration of an exemplary measurement system.

Fig. 14 includes an illustration of an exemplary measurement assembly.

Fig. 15 includes an illustration of an array of exemplary measurement components.

Fig. 16 includes an illustration of an exemplary pore structure.

Fig. 17 includes an illustration of an exemplary aperture and sensor configuration.

Fig. 18, 19, 20, and 21 include illustrations of work pieces during processing by exemplary methods.

Fig. 22, 23 and 24 include illustrations of work pieces during processing by exemplary methods.

Figures 25, 26 and 27 include illustrations of work pieces during processing by exemplary methods.

FIG. 28 shows an exemplary block diagram of components of a system for nucleic acid sequencing according to an exemplary embodiment.

Fig. 29 shows an exemplary cross-sectional view of a portion of an integrated circuit device and a flow cell in accordance with an exemplary embodiment.

FIG. 30 shows an exemplary cross-sectional view of a representative chemical sensor and corresponding reaction zone according to an exemplary embodiment.

Detailed Description

Conventional amplification of nucleic acid templates typically involves repeated replication of the template (and/or its progeny) using an appropriate synthesis system. In such conventional methods, each instance of replication typically begins by denaturing the template to be amplified using extreme denaturing conditions, thereby rendering the template substantially single-stranded. Some common and widely used examples of extreme denaturing conditions for conventional amplification include thermal denaturation using temperatures well above the melting temperature of the nucleic acid template to be amplified (e.g., conventional PCR includes thermal cycling using denaturation temperatures well above 90 ℃, typically about 94-95 ℃), or exposure of the template to powerful denaturing agents such as NaOH, guanidinium reagents, and the like. Such methods typically require specialized equipment (e.g., thermal cyclers) and additional operations (e.g., annealing steps for conventional PCR; washing steps for removal of chemical denaturants, etc.) during the amplification process, thereby increasing the costs, effort and time associated with such amplification and limiting the yields that can ultimately be obtained using such methods. Furthermore, such extreme denaturing conditions typically render the templates to be amplified substantially single-stranded, thereby presenting challenges for a number of applications involving multiplex clonal amplification (i.e., clonal amplification of a plurality of different templates within the same reaction mixture). For such multiplex applications, the use of these extreme denaturing conditions can be counterproductive, as this typically results in the release of one strand of the template from its associated site, allowing the released strand to migrate freely within solution and contaminate other amplicons that develop in close proximity. Such cross-contamination typically results in reduced yields of monoclonal amplified populations and increased yields of polyclonal contaminants (which are typically not available for many downstream applications). Improved nucleic acid amplification methods (and related compositions, systems, and kits) are needed to eliminate the drawbacks associated with conventional amplification methods.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification, including amplifying a nucleic acid template to produce an amplicon comprising a substantially monoclonal polynucleotide population, and related compositions, systems, and devices. Monoclonal properties are generally considered desirable in nucleic acid assays because the different properties of different polynucleotides within a polyclonal population can complicate interpretation of assay data. One example involves nucleic acid sequencing applications where the presence of polyclonal populations can complicate interpretation of sequencing data; however, many sequencing systems are inadequate to sense the detection of nucleotide sequence data from a single polynucleotide template, and thus require clonal amplification of the template prior to sequencing.

In some embodiments, the amplification methods of the present disclosure can be used to clonally amplify two or more different nucleic acid templates, optionally using the same reaction mixture and within the same reaction mixture, to produce at least two substantially monoclonal nucleic acid populations. Optionally, at least one substantially monoclonal population is formed by amplification of a single polynucleotide template.

Optionally, two or more different nucleic acid templates are amplified simultaneously and/or in parallel.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for nucleic acid synthesis, the methods comprising: providing at least two double-stranded nucleic acid templates in a reaction mixture; and clonally amplifying the at least two double-stranded nucleic acid templates according to any of the methods disclosed herein to form at least two substantially monoclonal nucleic acid populations.

In some embodiments, clonally amplifying optionally includes forming a reaction mixture. The reaction mixture may comprise a continuous liquid phase. In some embodiments, the continuous liquid phase comprises a single continuous aqueous phase. The liquid phase may comprise two or more polynucleotide templates, which may optionally have the same nucleotide sequence or may have nucleotide sequences that differ from each other. In some embodiments, at least one of the two or more polynucleotide templates may comprise at least one nucleic acid sequence that is substantially different or substantially non-complementary to at least one other polynucleotide template within the reaction mixture.

In some embodiments, two or more different nucleic acid templates are localized, placed, or located at different sites prior to amplification.

In some embodiments, two or more different nucleic acid templates are clonally amplified, optionally within a single reaction mixture, in solution, and after such clonal amplification, the resulting two or more substantially monoclonal nucleic acid populations are then localized, placed, or located at different sites.

The different sites are optionally members of an array of sites. The array may comprise a two-dimensional array of sites on a surface (e.g., the surface of a flow cell, electronic device, transistor chip, reaction chamber, tank, etc.) or a three-dimensional array of sites within a matrix or other vehicle (e.g., solid, semi-solid, liquid, fluid, etc.).

Optionally, two or more different nucleic acid templates are amplified within a continuous liquid phase, typically a continuous aqueous phase, of the same reaction mixture, thereby producing two or more different and substantially monoclonal polynucleotide populations, wherein each polynucleotide population is produced by amplification of a single polynucleotide template present in the reaction mixture.

Optionally, the continuous liquid phase is contained within a single phase or the same phase of the reaction mixture.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for nucleic acid synthesis, the methods comprising: providing a double-stranded nucleic acid template; and forming a population of substantially monoclonal nucleic acids by amplifying the double-stranded nucleic acid templates. Optionally, amplifying includes clonally amplifying the double-stranded nucleic acid template.

Optionally, amplifying comprises performing at least one amplification round under substantially isothermal conditions.

Optionally, amplifying comprises performing at least two consecutive nucleic acid synthesis cycles under substantially isothermal conditions.

In some embodiments, the amplification comprises Recombinase Polymerase Amplification (RPA). For example, the amplification may comprise performing at least one RPA round.

In some embodiments, the amplification includes template walking. For example, the amplification may include performing at least one template walking round.

In some embodiments, amplification optionally includes performing two different amplification rounds within the site or reaction chamber. For example, amplification may include performing at least one RPA round in any order or combination of rounds in a site or reaction chamber and at least one template walking round in a site or reaction chamber. In some embodiments, at least two consecutive cycles in any one or more amplification rounds are performed under substantially isothermal conditions. In some embodiments, at least one of the amplification rounds is performed under substantially isothermal conditions.

Optionally, the nucleic acid template to be amplified is double-stranded, or the template is rendered at least partially double-stranded prior to amplification using an appropriate procedure. (templates to be amplified are used interchangeably herein with nucleic acid templates or polynucleotide templates). In some embodiments, the template is linear. Alternatively, the template may be circular, or comprise a combination of linear and circular regions.

Optionally, the double-stranded nucleic acid template comprises a forward strand. The double-stranded nucleic acid template may also comprise a reverse strand. The forward strand optionally comprises a first primer binding site. The reverse strand optionally comprises a second primer binding site.

In some embodiments, the template already comprises a first and/or second primer binding site. Alternatively, the template optionally initially does not comprise a primer binding site, and the disclosed methods optionally include ligating or introducing the primer binding site to the template prior to amplification. For example, the method may optionally include ligating or otherwise introducing a linker comprising a primer binding site to the template. The linker may be attached to or otherwise introduced into the end of the linear template or the body of the linear or circular template. Optionally, the template may be cyclized upon ligation or after introduction of the linker. In some embodiments, a first linker may be attached to or introduced into a first end of a linear template and a second linker may be attached to or introduced into a second end of the template.

In some embodiments, amplifying comprises contacting the partially denatured template with a first primer, with a second primer, or with the first primer and the second primer in any order or combination.

In some embodiments, the first primer comprises a first primer sequence. The first primer optionally comprises an extendable terminus (e.g., a 3' OH-containing terminus). The first primer may optionally be attached to a compound (e.g. "drag tag") or to a support (e.g. a bead or site or surface of a reaction chamber).

In some embodiments, the second primer comprises a second primer sequence. The second primer optionally comprises an extendable terminus (e.g., a 3' OH-containing terminus). The second primer may optionally be attached to a compound (e.g., a "resistance tag") or to a support (e.g., a bead or site or surface of a reaction chamber).

Optionally, the first primer binds to the first primer binding site to form a first primer-template duplex. The second primer can bind to the second primer binding site to form a second primer-template duplex.

In some embodiments, amplifying includes extending the first primer to form an extended first primer. For example, amplifying can include extending a first primer of a first primer-template duplex to form an extended first primer.

Optionally, the extension is performed by a polymerase. The polymerase may be a strand displacement polymerase.

In some embodiments, amplifying comprises contacting the template to be amplified with a recombinase.

In some embodiments, amplifying comprises forming a partially denatured template. For example, amplification may include partially denaturing the double-stranded nucleic acid template.

Optionally, partially denaturing comprises subjecting the double-stranded nucleic acid template to partial denaturing conditions.

In some embodiments, the partial denaturation conditions include temperatures less than the Tm of the nucleic acid template, including, for example, temperatures 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ below the Tm of the nucleic acid template. In some embodiments, the partial denaturation conditions include a temperature above (e.g., at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ higher) the Tm of the first primer, the second primer, or the first and second primers. In some embodiments, the partial denaturation conditions include a temperature above (e.g., at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ higher) the Tm of the first primer binding site, the second primer binding site, or both the first primer binding site and the second primer binding site. In some embodiments, the nucleic acid template may comprise a linker sequence at one or both ends, and the partial denaturation conditions may include a temperature above the Tm of the linker sequence. In some embodiments, partial denaturation conditions (particularly partial denaturation temperatures) are used to selectively amplify nucleic acid templates during "template walking", as further described herein.

In other embodiments, the partial denaturing conditions comprise treating or contacting the nucleic acid template to be amplified with one or more enzymes capable of partially denaturing the nucleic acid template, optionally in a sequence-specific or sequence-oriented manner. In some embodiments, at least one enzyme optionally catalyzes strand invasion and/or unwinding in a sequence-specific manner. Optionally, the one or more enzymes comprise one or more enzymes selected from the group consisting of a recombinase, a topoisomerase, and a helicase. In some embodiments, partially denaturing the template may include contacting the template with a recombinase and forming a nucleoprotein complex comprising the recombinase. Optionally, contacting the template with a recombinase in the presence of the first primer, the second primer, or both the first and second primers. Partial denaturation can include the use of a recombinase to catalyze strand exchange and hybridization of a first primer to a first primer binding site (or hybridization of a second primer to a second primer binding site). In some embodiments, the partial denaturation includes strand exchange using a recombinase and hybridizing a first primer to a first primer binding site and a second primer to a second primer binding site.

In some embodiments, the partially denatured template comprises a single-stranded portion and a double-stranded portion. In some embodiments, the single stranded portion comprises a first primer binding site. In some embodiments, the single stranded portion comprises a second primer binding site. In some embodiments, the single stranded portion comprises a first primer binding site and a second primer binding site.

In some embodiments, partially denaturing the template includes contacting the template with one or more nucleoprotein complexes. At least one of the nucleoprotein complexes may comprise a recombinase. At least one of the nucleoprotein complexes may comprise a primer (e.g., a first primer or a second primer, or a primer comprising a sequence complementary to a corresponding primer binding sequence in the template). In some embodiments, partially denaturing the template may include contacting the template with a nucleoprotein complex containing primers. Partial denaturation can include hybridization of primers of the nucleoprotein complex to corresponding primer binding sites in the template, thereby forming primer-template double strands.

In some embodiments, partially denaturing the template may include contacting the template with a first nucleoprotein complex comprising a first primer. Partial denaturation can include hybridization of the first primer of the first nucleoprotein complex to the first primer binding site of the forward strand, thereby forming a first primer-template duplex.

In some embodiments, partially denaturing the template may include contacting the template with a second nucleoprotein complex comprising a second primer. Partial denaturation can include hybridization of the second primer of the second nucleoprotein complex to the second primer binding site of the reverse strand, thereby forming a second primer-template duplex.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise one or more primer extension steps. For example, the method may comprise extending the primer by incorporating nucleotides using a polymerase.

In some embodiments, the polymerase is a strand displacement polymerase.

Optionally, extending the primer comprises contacting the primer with a polymerase and one or more types of nucleotides under nucleotide incorporation conditions. In some embodiments, one or more types of nucleotides do not comprise an exogenous label, particularly an optically detectable label, such as a fluorescent moiety or dye. Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.). Typically, extension primers occur in a template dependent manner.

Optionally, the disclosed methods (and related compositions, systems, and kits) include extending the first primer by incorporating one or more nucleotides into the first primer of the first primer-template duplex using a polymerase, thereby forming an extended first primer.

Optionally, the disclosed methods (and related compositions, systems, and kits) include binding the second primer to the second primer binding site of the first extended primer by any suitable method (e.g., ligation or hybridization).

Optionally, the disclosed methods (and related compositions, systems, and kits) include extending the second primer by incorporating one or more nucleotides into the second primer of the second primer-template duplex using a polymerase, thereby forming an extended second primer.

In some embodiments, extending the first primer results in the formation of a first extended primer. The first extended primer may comprise some or all of the reverse strand sequence of the template. Optionally, the first extended primer comprises a second primer binding site.

In some embodiments, extending the second primer results in the formation of a second extended primer. The second extended primer may comprise some or all of the forward strand sequence of the template. Optionally, the second extended primer comprises a first primer binding site.

In some embodiments, the method is performed without subjecting the double-stranded nucleic acid template to extreme denaturing conditions during amplification. For example, the method can be performed without subjecting the nucleic acid template to a temperature greater than or equal to the Tm of the template during amplification. In some embodiments, the method can be performed without contacting the template with a chemical denaturant, such as NaOH, urea, guanidine salts, etc., during amplification. In some embodiments, amplifying comprises isothermal amplification.

In some embodiments, the method is performed without subjecting the nucleic acid template to extreme denaturing conditions during at least two, three, four, or more than four consecutive nucleic acid synthesis cycles. For example, the method may comprise two, three, four, or more than four consecutive nucleic acid synthesis cycles without contacting the nucleic acid template with a chemical denaturant. In some embodiments, the method may comprise performing two, three, four, or more than four consecutive nucleic acid synthesis cycles without subjecting the nucleic acid template to a

temperature

25, 20, 15, 10, 5, 2, or 1 ℃ above the actual or calculated Tm of the template or group of templates (or the actual or calculated average Tm of the template or group of templates). Two, three, four or more than four consecutive nucleic acid synthesis cycles may include intervening partial denaturation and/or primer extension steps.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise ligating one or more extended primer strands to the support. Ligation may optionally be performed during amplification or alternatively after amplification is complete. In some embodiments, the support comprises a plurality of second primers, and the method may comprise hybridizing at least one extended first primer strand to the second primers of the support.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise ligating one or more extended second primer strands to the support. In some embodiments, the support is attached to the first primer. For example, the support may comprise a plurality of first primers, and the method may comprise hybridizing at least one extended second primer to the first primer of the support, thereby ligating the extended second primer to the support. For example, a first primer may hybridize to a first primer binding site in an extended second primer.

In some embodiments, the support is attached to a second primer. For example, the support may comprise a plurality of second primers, and the method may comprise hybridizing at least one extended first primer to the second primers of the support, thereby ligating the extended first primers to the support. For example, a first primer may hybridize to a second primer binding site in an extended first primer.

In some embodiments, the support comprises at least one first primer and at least one second primer, and the disclosed methods (and related compositions, systems, and kits) comprise ligating the extended first primer and the extended second primer to the support.

Optionally, the support is attached to a target-specific primer. The target-specific primer optionally hybridizes (or is capable of hybridizing) to a first subset of templates within the reaction mixture, but does not bind to a second subset of templates within the reaction mixture.

Optionally, the support is attached to a universal primer. The universal primer optionally hybridizes (or is capable of hybridizing) to all or substantially all of the templates within the reaction mixture.

Optionally, the reaction mixture comprises a first support covalently linked to a first target-specific primer and a second support covalently linked to a second target-specific primer, and wherein the first and second target-specific primers are different from each other.

Optionally, the first target-specific primer is substantially complementary to the first target nucleic acid sequence and the second target-specific primer is substantially complementary to the second target nucleic acid sequence, and wherein the first and second target nucleic acid sequences are different.

In some embodiments, the disclosed methods include forming a first amplicon by amplifying a first template on a first support and forming a second amplicon by amplifying a second template on a second support, optionally within the same continuous phase of the reaction mixture. The first amplicon is optionally linked or attached to a first support and the second amplicon is optionally linked or attached to a second support.

The disclosed methods optionally include generating two or more monoclonal or substantially monoclonal amplicons by clonally amplifying two or more polynucleotide templates. Optionally clonally amplifying two or more polynucleotide templates within a continuous liquid phase of the amplification reaction mixture. The continuous liquid phase of the amplification reaction mixture may comprise a continuous aqueous phase. In some embodiments, amplifying comprises generating at least two substantially monoclonal amplified polynucleotide populations, each of which is formed by amplification of a single polynucleotide template. Optionally, clonally amplifying includes at least one RPA round. Optionally, clonally amplifying includes at least one template walking round.

In some embodiments, amplifying optionally includes forming an amplification reaction mixture comprising a continuous liquid phase. In some embodiments, the continuous liquid phase is a single continuous aqueous phase. The liquid phase may comprise two or more polynucleotide templates, which may optionally be different from each other. For example, two or more polynucleotide templates may comprise at least one nucleic acid sequence that is substantially different or substantially non-complementary to at least one other polynucleotide template within the amplification reaction mixture.

In some embodiments, amplifying optionally includes forming an amplification reaction mixture comprising a single continuous aqueous phase having two or more polynucleotide templates. Amplification optionally includes forming two or more substantially monoclonal nucleic acid populations by clonally amplifying two or more polynucleotide templates within a single aqueous phase. Optionally, clonally amplifying includes at least one RPA round. Optionally, clonally amplifying includes at least one template walking round.

In some embodiments, the present disclosure generally relates to methods (and related compositions, systems, and kits) for optionally amplifying one or more nucleic acid templates in parallel using partial denaturing conditions. In some embodiments, such methods are optionally used in an array format to amplify two or more templates. Optionally, the templates are amplified in bulk in solution prior to partitioning into the array. Alternatively, the template is first assigned to a site in the array, and then amplified in situ at (or within) the site of the array.

Optionally, the template is single-stranded or double-stranded. The template optionally comprises one or more primer binding sites.

In some embodiments, the method may include subjecting a double-stranded nucleic acid template comprising a primer binding site on at least one strand to at least one template-based replication cycle using a polymerase.

Optionally, the at least one template-based replication cycle comprises a partial denaturation step, an annealing step, and an extension step.

In some embodiments, the method comprises amplifying the double-stranded nucleic acid template by subjecting the template to at least two consecutive template-based replication cycles.

In some embodiments, the method comprises partially denaturing the template. Optionally, the method comprises forming a partially denatured template comprising single-stranded regions. The partially denatured template may also include a double stranded region. The single stranded region may comprise a primer binding site.

Optionally, partially denaturing comprises subjecting the template to a temperature at least 20, 15, 10, 5, 2, or 1 ℃ below the Tm of the primer binding site.

Optionally, partially denaturing comprises subjecting the template to a temperature at or above the Tm of the primer binding site.

Optionally, the partial denaturation includes contacting the double stranded template with a recombinase and a primer. The recombinase and primer may form part of a nucleoprotein complex, and the partial denaturation includes contacting the template with the complex.

In some embodiments, the method comprises forming a primer-template duplex by hybridizing a primer to a primer binding site of the single stranded region. In some embodiments, the template to be priming comprises a double stranded region. Optionally, the double stranded region does not comprise a primer binding site.

In some embodiments, the method comprises extending a primer of a primer-template duplex. Optionally, the method comprises forming an extended primer.

In some embodiments, different templates can be clonally amplified on different discrete supports (inflow beads or particles) without partitioning prior to amplification. In other embodiments, the template is partitioned or partitioned into an emulsion prior to amplification. Optionally, a template is dispensed into the droplet forming a portion of the hydrophilic phase of the emulsion having a discontinuous hydrophilic phase and a continuous hydrophobic phase. In some embodiments, the emulsion droplets of the hydrophilic phase further comprise one or more components necessary to perform PRA. For example, the emulsion droplets may comprise a recombinase enzyme. Optionally, the microdroplet comprises a strand displacement polymerase. In some embodiments, the microdroplet comprises a support immobilized primer and/or a solution phase primer. Optionally, the primer may bind to the template or to an amplification product thereof.

In some embodiments, the compositions, systems, methods, devices, and kits disclosed herein for nucleic acid amplification using emulsion-based amplification, including nucleic acid synthesis following partial denaturation of the template, provide advantageous aspects over conventional extension methods, including emulsion-based PCR or emPCR involving traditional thermal cycling. For example, nucleic acid amplification reactions including emulsion-based RPA ("emppa") or emulsion-based template walking can produce longer amplified polynucleotide templates, have fewer amplification steps, reduced time to prepare amplified polynucleotide templates, and/or increased sequencing data quality. Some suitable emulsion compositions for use with the amplification methods disclosed herein can be found, for example, in U.S. patent nos. 7622280, 7601499, and 7323305, which are incorporated by reference in their entirety.

In some embodiments, the method comprises providing a double-stranded template comprising a forward strand comprising a first primer binding site, a reverse strand comprising a second primer binding site; partially denaturing the template and forming a partially denatured template comprising a single-stranded region comprising the first primer binding site and at least one double-stranded region; forming a first primer-template duplex by hybridizing a first primer to a first primer binding site of the single-stranded region; extending the first primer of the first primer-template duplex using a polymerase to form an extended first primer comprising a second primer binding site, wherein the extended first primer hybridizes at least partially to the forward strand of the template; partially denaturing the extended first strand from the template to form a single stranded region comprising a second primer binding site; hybridizing the second primer to the second primer binding site of the single-stranded region and forming a second primer-template double strand, and extending the second primer of the second primer-template double strand, thereby forming an extended second primer.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for synthesizing nucleic acids from nucleic acid templates, comprising: providing a first nucleic acid duplex comprising a forward strand and a reverse strand, wherein the forward strand comprises a forward primer binding site and the reverse strand comprises a reverse primer binding site, and wherein the first duplex has a first melting temperature ("template Tm"), the forward primer binding site has a second melting temperature ("forward primer Tm"), and the reverse primer binding site has a third melting temperature ("reverse primer Tm"); partially denaturing the first double strand, wherein the partially denatured first double strand comprises a single strand region comprising a forward primer binding site and at least one double strand region; forming a first duplex to be priming by hybridizing the forward primer to the forward primer binding site of the partially denatured first duplex; extending the forward primer by contacting the first duplex to be primed with a strand displacement polymerase and a nucleotide under primer extension conditions, thereby forming a second duplex having a fourth melting temperature ("fourth Tm") comprising one strand comprising a forward primer binding site and one strand comprising a reverse primer binding site; partially denaturing the second double strand, wherein the partially denatured second double strand comprises a single stranded region comprising a reverse primer binding site and at least one double stranded region; forming a second duplex to be reverse priming by hybridizing the reverse primer to the reverse primer binding site of the partially denatured second duplex; extending the reverse primer of the reverse priming second duplex by contacting the reverse priming second duplex with a strand displacement polymerase and a nucleotide under primer extension conditions.

In some embodiments, the methods (and related compositions, systems, and kits) can further comprise sequencing the amplified template or sequencing the extended primer (e.g., the extended first primer or the extended second primer). Sequencing may include any suitable sequencing method known in the art. In some embodiments, sequencing includes sequencing by synthesis or sequencing by electronic detection (e.g., nanopore sequencing). In some embodiments, sequencing comprises extending a template or amplified template or extending a sequencing primer hybridized to a template or amplified template by polymerase-mediated nucleotide incorporation. In some embodiments, sequencing comprises sequencing a template or amplified template attached to a support by contacting the template or extended primer with a sequencing primer, a polymerase, and at least one type of nucleotide. In some embodiments, sequencing comprises contacting the template or amplified template or extended primer with a sequencing primer, a polymerase, and with only one type of nucleotide that does not contain an exogenous tag or chain terminating group.

Optionally, the template (or amplified product) may be placed, localized or located at a site. In some embodiments, the first primer of the plurality of templates/amplified templates/extensions is placed or located at a different site in the array of sites. In some embodiments, placement, localization, or localization is performed prior to template amplification. In some embodiments, placement, localization, or localization is performed after amplification. For example, the amplified template or extended first primer may be placed, located or localized at a different site of the array.

The methods disclosed herein result in the production of a plurality of amplicons, at least some of which comprise clonally amplified nucleic acid populations. Clonally amplified populations produced by the methods of the present disclosure can be used for a variety of purposes. In some embodiments, the disclosed methods (and related compositions, systems, and kits) optionally further comprise analysis and/or processing of clonally amplified populations (amplicons). For example, in some embodiments, the number of amplicons exhibiting certain desired characteristics may be detected and optionally quantified.

In some embodiments, the method may include determining whichThe discrete support (e.g., bead) comprises an amplicon. Similarly, the method may comprise determining which sites of the array comprise amplicons. DNA-based detection procedures such as UV absorbance, staining with DNA-specific dyes, and,

Assays, qPCR, hybridization with fluorescent probes, etc., to detect the presence of amplicons on the assay support or site. In some embodiments, the method may include determining which bead supports (or sites of the array) have obtained substantially monoclonal amplicons. For example, the bead supports (or array sites) may be analyzed to determine which supports or sites can produce a detectable and coherent (i.e., analyzable) sequence-dependent signal.

In some embodiments, amplification is followed by sequencing of the amplified product. Amplified products that are sequenced can include amplicons that comprise a substantially monoclonal population of nucleic acids. In some embodiments, the disclosed methods include forming or locating individual members of multiple amplicons at different sites. The different sites optionally form part of an array of sites. In some embodiments, the sites in the site array comprise wells (reaction chambers) on the surface of the isFET array.

In some embodiments, the method of downstream analysis comprises sequencing at least some of the plurality of amplicons in parallel. Optionally, the first primer of multiple templates/amplified templates/extensions at different array sites is sequenced in parallel.

In some embodiments, sequencing can include binding sequencing primers to nucleic acids of at least two different amplicons or at least two different substantially monoclonal populations.

In some embodiments, sequencing may include incorporating nucleotides into the sequencing primer using a polymerase. Optionally, incorporating includes forming at least one nucleotide incorporation byproduct.

Optionally, the nucleic acid to be sequenced is located at a site. The sites may include reaction chambers or wells. The sites may be part of an array of similar or identical sites. The array may comprise a two-dimensional array of sites on a surface (e.g., the surface of a flow cell, electronic device, transistor chip, reaction chamber, tank, etc.) or a three-dimensional array of sites within a matrix or other vehicle (e.g., solid, semi-solid, liquid, fluid, etc.).

In some embodiments, the site is operably coupled to a sensor. The method may include detecting nucleotide incorporation using a sensor. Optionally, the sites and the sensors are located in an array of sites coupled to the sensors.

In some embodiments, the sites comprise a hydrophilic polymer matrix conformally disposed within a well operably coupled to the sensor.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, the hydrophilic polymer matrix comprises polyacrylamide, a copolymer thereof, a derivative thereof, or a combination thereof.

Optionally, polyacrylamide is conjugated to the oligonucleotide primer.

Optionally, the pores have a characteristic diameter of 0.1 microns to 2 microns.

Optionally, the holes have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensor includes a Field Effect Transistor (FET). The FET may comprise an Ion Sensitive FET (ISFET).

In some embodiments, the methods (and related compositions, systems, and kits) can include detecting the presence of one or more nucleotide incorporation byproducts at the array site, optionally using FETs.

In some embodiments, the method may include optionally detecting a pH change occurring within the at least one reaction chamber using a FET.

In some embodiments, the disclosed methods include introducing a nucleotide into a site; and detecting an output signal from the sensor due to incorporation of the nucleotide into the sequencing primer. The output signal is optionally based on the threshold voltage of the FET. In some embodiments, the FET includes a floating gate conductor coupled to the sites.

In some embodiments, the FET includes a floating gate structure comprising a plurality of conductors electrically coupled to each other and separated by a dielectric layer, and the floating gate conductor is an uppermost conductor of the plurality of conductors.

In some embodiments, the floating gate conductor comprises an upper surface defining a bottom surface of the site.

In some embodiments, the floating gate conductor comprises a conductive material and the upper surface of the floating gate conductor comprises an oxide of the conductive material.

In some embodiments, the floating gate conductor is coupled to the at least one reaction chamber through a sensing material.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the amplification reaction mixture may comprise a recombinase. The recombinase may comprise any suitable agent that facilitates recombination between the polynucleotide molecules. The recombinase may be an enzyme that catalyzes homologous recombination. For example, the amplification reaction mixture may comprise a recombinase comprising or derived from a bacterial, eukaryotic or viral (e.g., phage) recombinase.

In some embodiments, the amplification reaction mixture comprises an enzyme that can bind the primer and the polynucleotide template to form a complex or can catalyze strand invasion of the polynucleotide template to form a D-ring structure. In some embodiments, the amplification reaction mixture comprises one or more proteins selected from the group consisting of UvsX, recA, and Rad 51.

In some embodiments, the amplification reaction mixture may comprise a recombinase-assisted protein such as UvsY.

In some embodiments, the amplification reaction mixture may comprise single-stranded binding proteins (SSBP).

In some embodiments, the amplification reaction mixture may comprise a polymerase. The polymerase optionally has or lacks exonuclease activity. In some embodiments, the polymerase has 5 'to 3' exonuclease activity, 3 'to 5' exonuclease activity, or both. Optionally, the polymerase lacks any one or more of such exonuclease activities.

In some embodiments, the polymerase has strand displacement activity.

In some embodiments, the amplification reaction mixture may comprise one or more solid or semi-solid supports. At least one of the supports may comprise one or more first primers comprising a first primer sequence. In some embodiments, at least one polynucleotide in the reaction mixture comprises a first primer binding sequence. The first primer binding sequence may be substantially identical or substantially complementary to the first primer sequence. In some embodiments, at least one, some, or all of the supports comprise a plurality of first primers that are substantially identical to each other. In some embodiments, all primers on the support are substantially identical to each other, or all primers comprise substantially identical first primer sequences.

In some embodiments, at least one support comprises two or more different primers attached thereto. For example, the at least one support may comprise at least one first primer and at least one second primer.

In some embodiments, the aqueous phase of the reaction mixture comprises a plurality of supports, wherein at least two supports of the plurality of supports are linked to a primer comprising a first primer sequence. In some embodiments, the reaction mixture comprises two or more different polynucleotide templates having a first primer binding sequence.

In some embodiments, the amplification reaction mixture may comprise a diffusion limiting agent. The diffusion limiting agent may be any agent effective to prevent or slow the diffusion of one or more of the polynucleotide templates and/or one or more of the amplification reaction products through the amplification reaction mixture.

In some embodiments, the amplification reaction mixture may comprise a sieving agent. The sieving agent may be any agent effective to sieving one or more polynucleotides (e.g., amplification reaction products and/or polynucleotide templates) present in the amplification reaction mixture. In some embodiments, the sieving agent limits or slows the migration of polynucleotide amplification products through the reaction mixture.

In some embodiments, the amplification reaction mixture may comprise crowding reagents.

In some embodiments, the amplification reaction mixture comprises a crowding reagent and a sieving reagent.

In some embodiments, the disclosed methods comprise clonally amplifying at least two of the two or more polynucleotide templates by: (a) Forming an amplification reaction mixture comprising a single continuous liquid phase comprising two or more polynucleotide templates, one or more surfaces or supports, and an amplification component; and (b) clonally amplifying at least two of the polynucleotide templates on one or more supports. Optionally, clonally amplifying includes forming at least two different substantially monoclonal amplicons. In some embodiments, clonally amplifying comprises subjecting the amplification reaction mixture to amplification conditions. In some embodiments, two or more of the amplicons are each attached to a surface or support. For example, the amplification reaction mixture may comprise a single support or surface such that each polynucleotide template is attached to a given region of the support or surface.

In some embodiments, the method for nucleic acid amplification may be performed in a single reaction vessel.

In some embodiments, the method for nucleic acid amplification may be performed in a single continuous liquid phase that does not provide for partitioning of multiple nucleic acid amplification reactions that occur in a single reaction vessel. In some embodiments, the method for nucleic acid amplification may be performed in a water-in-oil emulsion (microreaction vessel) that provides a partition.

In some embodiments, methods for nucleic acid amplification may be performed to attach a plurality of polynucleotides to a support or surface. For example, the method can include forming a reaction mixture comprising at least one surface, and subjecting the reaction mixture to amplification conditions. In some embodiments, the surface comprises a surface of a bead, a planar surface of a groove or tube, or an inner wall.

In some embodiments, a method for nucleic acid amplification comprises: (a) Forming an amplification reaction mixture comprising a single continuous liquid phase comprising a plurality of beads, a plurality of different polynucleotides, and a recombinase enzyme; (b) The amplification reaction mixture is subjected to isothermal amplification conditions, thereby producing a plurality of beads attached to a substantially monoclonal nucleic acid population attached thereto.

In some embodiments, the present disclosure generally relates to methods (and related compositions, systems, and kits) for array-based amplification of nucleic acid templates directly on the surface of the array, resulting in the formation of an array of any of its individual features, including amplicons (comprising a substantially monoclonal population of amplification products). These embodiments are in contrast to other embodiments described herein in which nucleic acid templates are optionally amplified in solution on discrete supports (e.g., beads) and then dispensed into an array.

In some embodiments, methods (and related compositions, systems, and kits) for array-based amplification are provided. In some embodiments, different polynucleotide templates are assigned to a site array and subsequently amplified in situ. The resulting amplicon array is then analyzed using an appropriate downstream procedure.

In some embodiments, a method for nucleic acid amplification comprises: a) Partitioning at least two different polynucleotides to a site by introducing a single polynucleotide into at least two sites in fluid communication with each other; and (b) forming at least two substantially monoclonal nucleic acid populations by amplifying the polynucleotides within the at least two sites. The sites may optionally include surfaces, wells, grooves, flow cells, reaction chambers, or channels. In some embodiments, amplification is performed without blocking the sites from each other. For example, at least two sites may be in fluid communication with each other during amplification.

In some embodiments, a method for nucleic acid amplification comprises: a) Partitioning at least two different polynucleotides to a site by introducing a single polynucleotide to at least two sites; and (b) forming at least two substantially monoclonal nucleic acid populations by amplifying the polynucleotides within the at least two sites. The sites may optionally include surfaces, wells, grooves, flow cells, reaction chambers, or channels. In some embodiments, amplification is performed without blocking the sites from each other. For example, at least two sites may be in fluid communication with each other during amplification.

In some embodiments, the site comprises a reaction chamber, and the method for nucleic acid amplification comprises: a) Partitioning at least two polynucleotide templates into reaction chambers by introducing a single polynucleotide into at least two reaction chambers that are in fluid communication with each other; and (b) forming at least two substantially monoclonal nucleic acid populations by amplifying the polynucleotide templates within the at least two reaction chambers. In some embodiments, amplification is performed without sealing the reaction chambers to each other. For example, at least two reaction chambers may be in fluid communication with each other during amplification.

In some embodiments, the site comprises a reaction chamber, and the method for nucleic acid amplification comprises: a) Partitioning at least two different polynucleotides into reaction chambers by introducing a single polynucleotide into at least two reaction chambers that are in fluid communication with each other; and (b) forming at least two substantially monoclonal nucleic acid populations by amplifying the polynucleotides within the at least two reaction chambers. In some embodiments, amplification is performed without sealing the reaction chambers to each other. For example, at least two reaction chambers may be in fluid communication with each other during amplification.

In some embodiments, the amplification steps of any and all methods of the present disclosure can be performed without completely denaturing the polynucleotide during amplification. For example, the disclosed methods can include amplifying at least two different polynucleotides by isothermal amplification. Amplification may include amplifying at least two different polynucleotides under substantially isothermal conditions. Optionally, the amplification is performed without contacting the polynucleotide with a chemical denaturing agent during the amplification.

In some embodiments, amplification optionally includes performing two different amplification rounds within a site or reaction chamber. For example, amplification may include performing at least one RPA round in any order or combination of rounds in a site or reaction chamber and at least one template walking round in a site or reaction chamber. In some embodiments, at least two consecutive cycles in any one or more amplification rounds are performed under substantially isothermal conditions. In some embodiments, at least one of the amplification rounds is performed under substantially isothermal conditions.

In some embodiments, amplifying comprises contacting the polynucleotide to be amplified with a reaction mixture. The contacting may optionally be performed before or after dispensing; it will be understood that the present disclosure includes embodiments in which polynucleotides are contacted with each component (or combination of components) of a reaction mixture at different times in succession, and embodiments in which any one or some components of a reaction mixture are contacted with at least two different polynucleotides prior to partitioning and the remaining components of a reaction mixture are contacted with at least two different polynucleotides after partitioning.

The at least two polynucleotides that are dispensed may optionally be used as templates for nucleic acid synthesis within their respective reaction chambers. In some embodiments, at least two polynucleotides comprise different sequences. In some embodiments, the polynucleotide is double-stranded or as single-stranded prior to partitioning. In some embodiments, the polynucleotide is linear, circular, or a combination of both. In typical embodiments, the polynucleotide is at least partially double-stranded (or dispensed in single-stranded form and then rendered at least partially double-stranded in a site or reaction chamber after dispensing). The polynucleotide may be made double-stranded prior to amplification (particularly in embodiments where amplification includes RPA or template walking).

In some embodiments, at least two different polynucleotide templates to be amplified each comprise a primer binding site, and amplifying comprises binding a primer to the primer binding site to form a primer-template duplex.

Optionally, amplifying includes extending the primer of the primer-template duplex. Extension optionally occurs at or within the sites or reaction chambers of the array. Optionally, extending the primer comprises contacting the primer with a polymerase and one or more types of nucleotides under nucleotide incorporation conditions. In some embodiments, one or more types of nucleotides do not comprise an exogenous label, particularly an optically detectable label, such as a fluorescent moiety or dye. Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.). Typically, extension primers occur in a template dependent manner.

Optionally, at least two different polynucleotides (i.e., templates to be amplified) each comprise a first sequence (referred to as a "first primer binding site") that is substantially identical or substantially complementary to at least some portion of the first primer.

In some embodiments, the reaction mixture includes a first primer comprising a first primer sequence. The first primer optionally comprises an extendable terminus (e.g., a 3' OH-containing terminus). The first primer may optionally be attached to a compound (e.g., a "resistance tag") or to a support (e.g., a bead or site or surface of a reaction chamber).

In some embodiments, at least two different polynucleotides comprise a second sequence that is substantially identical or substantially complementary to at least some portion of a second primer (referred to as a "second primer binding site").

In some embodiments, the method comprises extending the second primer by incorporating one or more nucleotides into the second primer of the second primer-template duplex using a polymerase, thereby forming an extended second primer.

In some embodiments, the method is performed without subjecting the nucleic acid template to extreme denaturing conditions during at least two, three, four, or more than four consecutive nucleic acid synthesis cycles. For example, the method may comprise two, three, four, or more than four consecutive nucleic acid synthesis steps without contacting the nucleic acid template with a chemical denaturing agent. In some embodiments, the method may comprise performing two, three, four, or more than four consecutive nucleic acid synthesis cycles without subjecting the nucleic acid template to a

temperature

In some embodiments, the disclosed methods (and related compositions, systems, and kits) can further comprise ligating one or more extended second primer strands to the support. In some embodiments, the support is attached to the first primer. For example, the support may comprise a plurality of first primers, and the method may comprise hybridizing at least one extended second primer to the first primer of the support, thereby ligating the extended second primer to the support. For example, a first primer may hybridize to a first primer binding site in an extended second primer. The support may comprise, for example, any array of surfaces.

In some embodiments, the disclosed methods include forming a first amplicon by amplifying a first template on a first support and a second amplicon by amplifying a second template on a second support, optionally within the same continuous phase of the reaction mixture and at different sites on a surface (e.g., within an array). The first amplicon is optionally linked or attached to a first support and the second amplicon is optionally linked or attached to a second support.

The disclosed methods optionally include generating two or more monoclonal or substantially monoclonal amplicons at two or more different sites of the array of sites by clonally amplifying the two or more polynucleotide templates, so as to form at least two sites to each comprise a substantially monoclonal nucleic acid population. Two or more polynucleotide templates are optionally placed or located at different sites and subsequently clonally amplified within a continuous liquid phase of an amplification reaction mixture that is in contact with the array. The continuous liquid phase of the amplification reaction mixture may comprise a continuous aqueous phase.

In some embodiments, amplifying comprises generating at least two substantially monoclonal amplified polynucleotide populations, each of which is formed by amplification of a single polynucleotide template.

Optionally, clonally amplifying includes at least one RPA round.

Optionally, clonally amplifying includes at least one template walking round.

In some embodiments, a plurality of different polynucleotide templates are placed or located at different sites prior to amplification. For example, the amplified template or extended first primer may be placed, located or localized at a different site of the array.

In some embodiments, amplification results in the formation of at least two substantially monoclonal nucleic acid populations (e.g., amplicons) in at least two different sites of a surface, which can then be analyzed in situ using an appropriate procedure.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for preparing a surface. Optionally, the surface comprises a plurality of sites comprising a first site and a second site.

In some embodiments, the method comprises forming an array of nucleic acids on the surface, wherein the forming comprises ligating a first nucleic acid to a first site and ligating a second nucleic acid to a second site. The ligation may optionally be performed by using any of the methods disclosed herein, including, for example, by ligating the nucleic acid to a primer that is covalently attached to the surface.

In some embodiments, the method comprises contacting at least the first and second nucleic acids with a single reaction mixture comprising reagents for nucleic acid synthesis. The reaction mixture may optionally comprise any one or more of the components described herein. In some embodiments, the reaction mixture comprises all components necessary to perform RPA. In some embodiments, the reaction mixture contains all components that perform template walking.

In some embodiments, the method comprises forming a first amplicon at a first locus and a second amplicon at a second locus by replicating the first or second nucleic acid using reagents for nucleic acid synthesis in the reaction mixture. Replication may include primer extension. Replication may include one or more RPA loops. Replication may include one or more template walking cycles.

In some embodiments, replication includes at least one RPA cycle.

In some embodiments, replication includes at least one template walking cycle.

In some embodiments, replication includes at least one RPA cycle and at least one template walking cycle.

In some embodiments, replication includes at least one RPA round.

In some embodiments, replication includes at least one template walking round.

In some embodiments, replication includes at least one RPA round and at least one template walking round.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for preparing a surface, the methods comprising: (a) Providing a surface having a plurality of sites comprising a first site and a second site; (b) Forming an array of nucleic acids on a surface, wherein the forming comprises ligating a first nucleic acid to a first site and ligating a second nucleic acid to a second site; (c); and (d) forming a first amplicon at the first site and a second amplicon at the second site by replicating the first or second nucleic acid using reagents for nucleic acid synthesis in the reaction mixture.

In some embodiments, the present disclosure generally relates to a method for preparing a surface, the method comprising: providing a surface having a plurality of sites comprising a first site and a second site; forming an array of nucleic acids on a surface, wherein the forming comprises ligating a first nucleic acid to a first site and ligating a second nucleic acid to a second site; contacting at least a first and a second nucleic acid with a single reaction mixture comprising reagents for nucleic acid synthesis; and forming a first substantially monoclonal amplicon at the first site and a second substantially monoclonal amplicon at the second site by amplifying the first or second nucleic acid using reagents for nucleic acid synthesis in the reaction mixture. Optionally, the first and second sites remain in fluid communication during amplification. Optionally, amplification is performed without complete denaturation of the polynucleotide during amplification. For example, the disclosed methods can include amplifying at least two different polynucleotides by isothermal amplification. Amplification may include amplifying at least two different polynucleotides under substantially isothermal conditions. Optionally, the amplification is performed without contacting the polynucleotide with a chemical denaturing agent during the amplification.

In some embodiments, at least one of the plurality of sites comprises a reaction well, groove, or chamber.

In some embodiments, at least one of the plurality of sites is connected to a sensor.

In some embodiments, the sensor is capable of detecting nucleotide incorporation occurring at or near at least one site.

In some embodiments, the sensor includes a Field Effect Transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites comprise a primer attached to the surface.

In some embodiments, at least one of the plurality of sites comprises a hydrophilic polymer matrix conformally disposed within a well operably coupled to the sensor.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, polyacrylamide is conjugated to the oligonucleotide primer.

Optionally, the holes have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensor includes a Field Effect Transistor (FET). The FETs may include Ion Sensitive FETs (ISFETs), chemFET, bioFET, etc.

In some embodiments, the FET is capable of detecting the presence of nucleotide incorporation byproducts at least one site.

In some embodiments, the FET is capable of detecting a chemical moiety selected from hydrogen ions, pyrophosphates, hydroxyl ions, and the like.

In some embodiments, the method can include optionally detecting a pH change occurring at the site or within the at least one reaction chamber using a FET.

In some embodiments, the disclosed methods comprise introducing a nucleotide into at least one of the plurality of sites; and detecting an output signal from the sensor due to incorporation of the nucleotide into the sequencing primer. The output signal is optionally based on the threshold voltage of the FET. In some embodiments, the FET includes a floating gate conductor coupled to the sites.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture comprises all components necessary to perform RPA.

In some embodiments, the reaction mixture comprises all components necessary to perform template walking.

In some embodiments, the reaction mixture may comprise one or more solid or semi-solid supports. At least one of the supports may comprise one or more first primers comprising a first primer sequence. In some embodiments, at least one of the supports comprises two or more different primers attached thereto. For example, the at least one support may comprise at least one first primer and at least one second primer.

Alternatively, in some embodiments, the reaction mixture does not comprise any support. In some embodiments, at least two different polynucleotide templates are amplified directly on the surface of the reaction chamber or the sites of the array.

In some embodiments, the reaction mixture may comprise a recombinase. The recombinase may comprise any suitable agent that facilitates recombination between the polynucleotide molecules. The recombinase may be an enzyme that catalyzes homologous recombination. For example, the reaction mixture may comprise a recombinase comprising or derived from a bacterial, eukaryotic or viral (e.g., phage) recombinase.

Optionally, the reaction mixture comprises nucleotides that are not exogenously labeled. For example, the nucleotide may be a naturally occurring nucleotide, or a synthetic analog that does not contain a fluorescent moiety, dye, or other exogenous optically detectable label. Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.).

Optionally, the reaction mixture comprises nucleotides, which are naturally occurring nucleotides. Optionally, the nucleotide does not contain a group that terminates nucleic acid synthesis (e.g., a dideoxy group, a reversible terminator, etc.).

In some embodiments, the reaction mixture comprises an enzyme that can bind the primer and the polynucleotide template to form a complex or can catalyze strand invasion of the polynucleotide template to form a D-ring structure. In some embodiments, the reaction mixture comprises one or more proteins selected from UvsX, recA, and Rad 51.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, and kits) for preparing a surface, the methods comprising: (a) Providing a surface having a plurality of sites, wherein each site is linked to a nucleic acid primer; (c) Contacting the surface with a plurality of polynucleotide templates and ligating at least one template to the surface; and amplifying at least one template on the surface, thereby forming a population of substantially monoclonal amplified target polynucleotide sequences at least one site on the surface.

In some embodiments, the disclosure generally relates to methods for nucleic acid amplification, comprising: (1) Providing a surface having a first site and a second site, the first site operably coupled to a first sensor and comprising a first template; a second site operably coupled to a second sensor and comprising a second template; (2) partitioning the reaction mixture to first and second sites; and (3) forming a first amplicon by amplifying the first template at the first site; and forming a second amplicon by amplifying the second template at the second locus.

In some embodiments, the amplification comprises at least one RPA cycle.

In some embodiments, the amplification comprises at least one template walking cycle.

In some embodiments, the amplification comprises at least one RPA cycle and at least one template walking cycle.

In some embodiments, the amplification comprises at least one RPA round.

In some embodiments, the amplification comprises at least one template walking round.

In some embodiments, the amplification comprises at least one RPA round and at least one template walking round.

In some embodiments, any or all of the methods disclosed herein can result in the production of a plurality of amplicons, at least some of which comprise a clonally amplified nucleic acid population. Clonally amplified populations produced by the methods of the present disclosure can be used for a variety of purposes. In some embodiments, the disclosed methods (and related compositions, systems, and kits) optionally further comprise analysis and/or processing of clonally amplified populations (amplicons).

In some embodiments, amplicons produced according to the present disclosure may be subjected to downstream analytical methods, such as sequencing.

In some embodiments, the amplified nucleic acid can be further analyzed (e.g., sequenced) at the assigned sites without recovering and moving the amplified product to a different site or surface for analysis (e.g., sequencing).

In some embodiments, the method of downstream analysis comprises sequencing at least a portion of the plurality of amplicons in parallel. Optionally, the first primer of multiple templates/amplified templates/extensions at different array sites is sequenced in parallel.

For example, in some embodiments, the amplified product is sequenced in situ after amplification. Amplified products that are sequenced can include amplicons that comprise a substantially monoclonal population of nucleic acids. Optionally, a population of monoclonal nucleic acids (amplicons) located at different sites of the array are sequenced in parallel.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, polyacrylamide is conjugated to the oligonucleotide primer.

Optionally, the holes have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the reaction mixture may comprise a recombinase helper protein such as UvsY.

In some embodiments, the reaction mixture may comprise a single chain binding protein (SSBP).

In some embodiments, the reaction mixture may comprise a polymerase. The polymerase optionally has or lacks exonuclease activity. In some embodiments, the polymerase has 5 'to 3' exonuclease activity, 3 'to 5' exonuclease activity, or both. Optionally, the polymerase lacks any one or more of such exonuclease activities.

In some embodiments, the polymerase has strand displacement activity.

In some embodiments, the reaction mixture may include a diffusion limiting agent. The diffusion limiting agent may be any agent effective to prevent or slow the diffusion of one or more of the polynucleotide templates and/or one or more of the amplification reaction products through the reaction mixture.

In some embodiments, the reaction mixture may comprise a sieving agent. The sieving agent may be any agent effective to sieving one or more polynucleotides (e.g., amplification reaction products and/or polynucleotide templates) present in the reaction mixture. In some embodiments, the sieving agent limits or slows the migration of polynucleotide amplification products through the reaction mixture.

In some embodiments, the reaction mixture may comprise crowding reagents.

In some embodiments, the reaction mixture comprises a crowding agent and a sieving agent.

In some embodiments, the disclosed methods comprise contacting each of the at least two polynucleotides with a recombinase enzyme, a support having a plurality of first oligonucleotide primers attached thereto (the first oligonucleotide primers being at least partially complementary to at least some portion of the polynucleotides), a polymerase, and the plurality of nucleotides in any order and in any combination.

In some embodiments, the at least two different polynucleotides comprise a forward strand comprising a first primer binding site, and the amplification within the at least two sites (or within the at least two reaction chambers) optionally comprises binding the first primer to the first primer binding site within the site or reaction chamber to form a first primer-template duplex. Optionally, binding of the first primer to at least two different polynucleotide templates is mediated by a recombinase. For example, amplification may include forming a nucleoprotein complex comprising a recombinase and a first primer. Optionally, the first primer is attached to the surface of the site or reaction chamber. In some embodiments, amplification in a site or reaction chamber comprises: a first nucleoprotein complex (or "first nucleoprotein filament") is formed. Amplification optionally further comprises contacting at least one of the polynucleotides in the site or reaction chamber with the first nucleoprotein filament, the polymerase and the plurality of nucleotides in any order or combination.

Optionally, each of the discrete supports (e.g., beads) comprising the plurality of first primers is dispensed into a reaction chamber or site prior to amplification, and amplifying comprises amplifying one of the at least two different polynucleotides on the support within the site or reaction chamber. In some embodiments, any of the partitioning and/or contacting steps may be repeated prior to amplification, optionally to increase the number and/or yield of sites or reaction chambers where monoclonal products are produced.

Optionally, amplifying includes extending the first primer of the first primer-template duplex within the reaction chamber using a polymerase, thereby forming an extended first primer. Optionally, extending the first primer replaces the reverse strand from the forward strand. The extended first primer optionally comprises a second primer binding site.

Optionally, the amplifying comprises a reverse synthesis step comprising binding the second primer to the second primer binding site of the extended first primer and extending the second primer to form a second primer-template duplex. Optionally, binding of the second primer to the polynucleotide template is mediated by a recombinase. For example, amplification may include forming a nucleoprotein complex comprising a recombinase and a second primer. Optionally, the second primer is attached to the surface of the site or reaction chamber. In some embodiments, amplification in a site or reaction chamber comprises: a second nucleoprotein complex (or "second nucleoprotein filament") is formed. Amplification optionally further comprises contacting at least one of the site or the polynucleotide template in the reaction chamber or at least one of the extended first primer with the second nucleoprotein filament, the polymerase and the plurality of nucleotides in any order or combination.

Optionally, amplifying comprises extending the first primer-template duplex, the second primer-template duplex, or both using a polymerase. The polymerase may have strand displacement activity.

In some embodiments, the methods (and related compositions, systems, and kits) can include placing, locating, or localizing at least one substantially monoclonal population at a site. The sites may form part of an array of sites.

Optionally, at least one of the sites comprises a reaction chamber, a support, a particle, a microparticle, a sphere, a bead, a filter, a flow cell, a well, a trench, a groove receptacle (channel reservoir), a gel, or an inner wall of a tube.

In some embodiments, at least one site comprises a hydrophilic polymer matrix conformally disposed within a well operably coupled to a sensor.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, polyacrylamide is conjugated to the oligonucleotide primer.

Optionally, the holes have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

Optionally, the plurality of different polynucleotide templates (or amplified polynucleotides) comprises at least one nucleic acid comprising a selectively cleavable moiety.

Optionally, the selectively cleavable moiety comprises uracil.

Optionally, the method for nucleic acid amplification further comprises cleaving the cleavable moiety with a cleavage agent.

Optionally, cleavage may be performed prior to amplification, for example, prior to formation of the reaction mixture.

Optionally, cleavage may be performed after amplification, e.g., after the nucleic acid template is amplified.

Optionally, the reaction mixture comprises at least one primer comprising a cleavable moiety.

Optionally, the plurality of different polynucleotides comprises a plurality of amplicons.

Optionally, the plurality of different polynucleotides comprises a plurality of different amplicons.

In some embodiments, amplification can be performed using any of the methods, compositions, systems, and kits disclosed in U.S. provisional patent application No. 61/792247 filed on 3, 15 (incorporated herein by reference in its entirety).

In some embodiments, amplicons produced according to the disclosure may be subjected to downstream analytical methods, such as quantification. For example, in some embodiments, the number of amplicons exhibiting certain desired characteristics may be detected and optionally quantified.

In some embodiments, the amplified nucleic acid may optionally be subjected to additional downstream analytical steps in the disclosed methods.

In some embodiments, including amplifying different polynucleotide templates on discrete and separate supports, the method may include determining which discrete supports (e.g., beads) comprise amplicons. Similarly, in embodiments in which templates are assigned to the array prior to amplification, the method may include determining which sites of the array contain amplicons, and optionally also counting the number of sites that contain amplicons. DNA-based detection procedures such as UV absorbance, staining with DNA-specific dyes, and,

Assays, qPCR, hybridization with fluorescent probes, etc., to detect the presence of amplicons on the assay support or site. In some embodiments, the method may include determining which bead supports (or sites of the array) have obtained substantially monoclonal amplicons. For example, the bead supports (or array sites) may be analyzed to determine which supports or sites may produce a detectable and coherent (i.e., analyzable) sequence dependent signal.

In some embodiments, the disclosed methods include additional downstream analysis steps that provide the same type of information previously obtained by conventional techniques such as digital PCR or digital RPA (as described, for example, in Shen 2011Analytical Chemistry 83:3533-3540; published U.S. patent applications US2012/0264132 and 2012/0329038 (all incorporated herein by reference in their entirety). Digital PCR (dPCR) is a modification of conventional Polymerase Chain Reaction (PCR) that can be used to directly quantify and clonally amplify nucleic acids, including DNA, cDNA, methylated DNA, or RNA. One difference between dPCR and traditional PCR is the method of measuring the amount of nucleic acid. PCR and dPCR each perform one reaction per single sample, dPCR also performs a single reaction within a sample, however the sample is divided into a large number of partitions and the reactions are performed individually in each partition. The separation allows for sensitive measurement of the amount of nucleic acid. dPCR has proven useful for studying changes in gene sequences, such as copy number changes or point mutations.

In contrast to the present method, dPCR typically requires partitioning of the sample prior to amplification; in contrast, several embodiments disclosed herein provide for parallel amplification of different templates within a single continuous phase of a reaction mixture without the need for zoning. In dPCR, the sample is typically partitioned so that individual nucleic acid molecules within the sample are localized and localized to a number of separate regions. The sample is partitioned by a simple dilution method so that each portion contains about 1 copy of the DNA template or less. By isolating individual DNA templates, the method effectively enriches DNA molecules present at very low levels in the original sample. The partitioning of the samples facilitates molecular counting using Poisson statistics. As a result, each partition will contain a "0" or "1" molecule, or a negative or positive reaction, respectively. While the initial copy number of a molecule is proportional to the number of amplification cycles in conventional PCR, dPCR generally does not depend on the number of amplification cycles to determine the initial sample size.

Conventional methods of dPCR analysis typically utilize fluorescent exploration and light-based detection methods to identify amplified products. Such methods require sufficient amplification of the target molecule to produce a sufficient detectable signal, but may result in additional errors or deviations.

In those embodiments of the disclosure that include the distribution of nucleic acid templates into wells of an isFET array and subsequent amplification of the templates in the wells of the array, an optional downstream analysis step may be performed after amplification that quantifies the number of sites or wells that contain amplification products. In some embodiments, the products of the nucleic acid amplification reaction can be detected to count the number of sites or wells containing amplified templates.

For example, in some embodiments, the disclosure generally relates to methods of nucleic acid synthesis comprising: providing a sample comprising a first amount of polynucleotides; and assigning individual polynucleotides of the sample to different sites of the array of sites.

Optionally, the method may further comprise forming a substantially monoclonal population of nucleic acids within their respective sites by amplifying the individual polynucleotides.

Optionally, the sites remain in fluid communication during amplification.

Optionally, the amplifying comprises partially denaturing the template.

Optionally, amplifying comprises subjecting the template to a partial denaturation temperature. In some embodiments, the template includes a low melting point sequence comprising a primer binding site that is rendered single stranded when the template is subjected to a partial denaturation temperature.

Optionally, the amplifying comprises partially denaturing the template.

Optionally, amplifying comprises contacting at least two different templates at two different array sites with a single reaction mixture for nucleic acid amplification.

Optionally, the reaction mixture comprises a recombinase.

Optionally, the reaction mixture comprises at least one primer comprising a "resistance tag".

Optionally, amplifying comprises performing at least one amplification cycle comprising: partially denaturing the template, hybridizing the primer to the template, and extending the primer in a template-dependent manner. Optionally, amplifying comprises isothermal amplification. In some embodiments, the amplification is performed under substantially isothermal conditions.

In some embodiments, the percentage of sites containing one or more template molecules is greater than 50% and less than 100%.

In some embodiments, the disclosed methods can further comprise detecting a change in ion concentration in the at least one site due to the at least one amplification cycle.

In some embodiments, the disclosed methods can further comprise quantifying the initial amount of the target nucleic acid.

Some examples of array-based digital PCR using ion-based sensing techniques can be found in, for example, U.S. provisional application No. 61/635584 (incorporated herein by reference in its entirety) filed on, for example, 4/19.

In some embodiments, the disclosure generally relates to methods for detecting a target nucleic acid, comprising: dividing the sample into a plurality of sample volumes, wherein more than 50% of the fractions comprise no more than 1 target nucleic acid molecule per sample volume; subjecting a plurality of sample volumes to conditions for amplification, wherein the conditions comprise partially denaturing conditions; detecting a change in ion concentration in a sample volume in which the target nucleic acid is present; counting the number of portions with amplified target nucleic acid; and determining the amount of target nucleic acid in the sample. The change in ion concentration may be an increase in ion concentration or may be a decrease in ion concentration. In some embodiments, the method may further comprise combining the sample with a bead. In some embodiments, a method may include loading a sample onto a substrate, wherein the substrate includes at least one aperture.

In some embodiments, subjecting the target nucleic acid to partial denaturing conditions comprises contacting the target nucleic acid molecules in their respective sample volumes with a recombinase and a polymerase under RPA conditions.

In some embodiments, subjecting the target nucleic acid to partial denaturation conditions comprises subjecting the target nucleic acid molecule to partial denaturation temperatures.

In some embodiments, the present disclosure relates generally to methods for performing absolute quantification of nucleic acids, comprising: diluting a sample comprising an initial amount of nucleic acid templates and partitioning the nucleic acid templates of the sample into a plurality of sites of an array, wherein the percentage of sites comprising one or more nucleic acid templates is greater than 50% and less than 100%; subjecting a plurality of sites to at least one amplification cycle, wherein the amplification cycle is performed according to any of the amplification methods disclosed herein; detecting a change in ion concentration in at least one of the plurality of sample volumes due to at least one amplification cycle; and quantifying the initial amount of nucleic acid template. The change in ion concentration may be an increase in ion concentration, a decrease in ion concentration, a change in pH, and may involve detection of positive ions such as hydrogen ions, anions such as pyrophosphate molecules, or cations and anions.

In some embodiments, the present disclosure also generally relates to methods (and related compositions, systems, and kits) for connecting individual members of a population of nucleic acid templates to different ones of a plurality of supports or to different ones of a plurality of sites by using recombinase-mediated strand exchange. These methods, compositions, systems and kits can be used to generate immobilized populations of amplicons suitable for manipulation in applications where it is desirable to obtain or differentiate different amplicons individually. In some embodiments, the plurality of discrete supports or sites in the array each comprise a capture primer. Immobilization of each template to each support (or to each site of the array) may be achieved by contacting the template with the support or site in the presence of a primer ("fusion primer"). In some embodiments, the fusion primer comprises a target-specific portion that is complementary to a portion of the template, and a universal primer binding site that is complementary to at least some portion of the capture primer of the support or site. Optionally, contacting is performed in the presence of an RPA component. The RPA component may comprise a recombinase enzyme. The RPA component may comprise a strand displacement polymerase. In some embodiments, the fusion primer is recombined into the template by recombinase-mediated strand exchange to form a template comprising a universal primer binding site, a primer adduct. In some embodiments, the capture primer is then recombined into the universal primer binding site, forming an immobilized template attached to a support or site.

In some embodiments of bead-based amplification, a library of fusion primers each comprising a different target-specific moiety and a common universal primer binding site is contacted with a plurality of templates and a plurality of supports in a reaction mixture comprising a polymerase and a strand displacement polymerase. The template library is then linked to a plurality of supports by subjecting the mixture to RPA conditions, thereby producing a plurality of supports each having a different template attached thereto.

In some embodiments of array-based amplification, a library of fusion primers each comprising a different target-specific moiety and a common universal primer binding site is contacted with a plurality of templates and a surface comprising a plurality of sites in a reaction mixture comprising a polymerase and a strand displacement polymerase. At least some of the plurality of sites comprise universal capture primers. The template library is then ligated to multiple sites on the surface by subjecting the mixture to RPA conditions, thereby producing multiple supports each having a different template attached.

In some embodiments, the disclosure generally relates to compositions (and related methods for making and using the compositions) comprising reagents for amplifying one or more nucleic acid templates in parallel using partial denaturing conditions.

In some embodiments, the composition may comprise any of the components described herein for performing RPA.

In some embodiments, the composition may comprise any of the components described herein for performing template walking.

In some embodiments, the present disclosure relates generally to compositions and systems for nucleic acid amplification, comprising: a surface comprising a first site and a second site; and a nucleic acid amplification reaction mixture, wherein the mixture is contacted with the first and second sites.

In some embodiments, the reaction mixture comprises a recombinase.

In some embodiments, the first site is operably coupled to the first sensor and the second site is operably connected to the second sensor.

In some embodiments, the first and second sites are operably linked to the same sensor.

Optionally, the first locus comprises a first population of substantially monoclonal nucleic acids. The second locus optionally comprises a second substantially monoclonal nucleic acid population.

In some embodiments, the disclosed compositions comprise: a surface comprising a first site and a second site, wherein the first site comprises a first substantially monoclonal nucleic acid population and the second site comprises a second substantially monoclonal nucleic acid population; and a nucleic acid amplification reaction mixture, wherein the mixture is contacted with the first and second sites.

In some embodiments, the composition comprises an array of sites comprising a first site comprising (e.g., linked to) a first capture primer and a second site comprising (e.g., linked to) a second capture primer.

In some embodiments, the sensor includes a Field Effect Transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites comprise a capture primer attached to the surface.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, polyacrylamide is conjugated to the oligonucleotide primer.

Optionally, the holes have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

In some embodiments, the method of amplification may include performing "template walking" as described in U.S. patent publication No. 2012/0156728 (which is incorporated herein by reference in its entirety) published at 21, 6, 2012. For example, in some embodiments, the present disclosure generally relates to methods, compositions, systems, devices, and kits for clonally amplifying one or more nucleic acid templates to form a clonally amplified population of nucleic acid templates. Any of the amplification methods described herein optionally include repeated nucleic acid amplification cycles. The amplification cycle optionally includes: (a) hybridization of the primer to the template strand, (b) primer extension to form a first extended strand, (c) partial or incomplete denaturation of the extended strand from the template strand. Primers that hybridize to the template strand (for convenience, designated "forward" primers) are optionally immobilized on or to a support. The support is, for example, solid or semi-solid. Optionally, the denatured portion of the template strand from step (c) is free to hybridize to a different forward primer in the next amplification cycle. In embodiments, primer extension in subsequent amplification cycles includes displacement of the first extended strand from the template strand. A second "reverse" primer may be included, for example, that hybridizes to the 3' end of the first extended strand. The reverse primer is optionally non-immobilized.

In embodiments, the template is amplified using primers immobilized on or to one or more solid or semi-solid supports. Optionally the support comprises immobilized primers complementary to the first portion of the template strand. Optionally, the support does not significantly comprise immobilized primers homologous to a second non-overlapping portion of the same template strand. Two parts are non-overlapping if they do not contain any sub-parts that hybridize to each other or to their complements. In another example, the support optionally does not significantly comprise immobilized primers that can hybridize to the complement of the template strand.

Optionally, in the presence of one or more supports in a single continuous liquid phase, wherein each support comprises one or more immobilization sites, a plurality of nucleic acid templates are simultaneously amplified. In embodiments, each template is amplified to produce clonal amplified populations, wherein each clonal population is immobilized in or on a different support or immobilization site than the other amplified populations. Optionally, the amplified population remains substantially monoclonal after amplification.

The template is, for example, amplified to produce a clonal population comprising template homology strands (referred to herein as "template strands" or "reverse strands") and/or template complementarity strands (referred to herein as "primer strands" or "forward strands"). In embodiments, clonality is maintained in the resulting amplified nucleic acid population by maintaining association between the template strand and its primer strand, thereby effectively associating or "tethering" related clonal progeny together and reducing the likelihood of cross-contamination between different clonal populations. Optionally, one or more amplified nucleic acids in the clonal population are attached to a support. The clonal population of substantially identical nucleic acids may optionally have spatially localized or discontinuous macroscopic manifestations. In embodiments, the clonal population may resemble isolated spots or colonies (e.g., optionally on an outer surface of a support when dispensed into the support).

In some embodiments, the present disclosure generally relates to novel methods of producing a localized clonal population of clonal amplicons, optionally immobilized to or in or on one or more supports. The support may be, for example, a solid or semi-solid (e.g., a gel or hydrogel). The amplified clonal population is optionally attached to the outer surface of the support or may also be within the inner surface of the support (e.g., when the support has a porous or matrix structure).

In some embodiments, amplification is achieved by multiple cycles of primer extension along the template strand of interest (also referred to as the "reverse" strand). For convenience, the primer that hybridizes to the template strand of interest is referred to as a "forward" primer, and is optionally extended in a template-dependent manner to form a "forward" strand that is complementary to the template strand of interest. In some methods, the forward strand itself hybridizes to a second primer, referred to as a "reverse" primer, which is extended to form a new template strand (also referred to as a reverse strand). Optionally, at least a portion of the new template strand is homologous to the original template of interest ("reverse") strand.

As mentioned, one or more primers may be immobilized to or in or on one or more supports. Optionally, one primer is immobilized by ligation to a support. The second primer may be present and optionally not immobilized or attached to a support. Different templates may be amplified simultaneously, e.g., in a single continuous liquid phase, on different supports or immobilization sites to form a population of monoclonal nucleic acids. A liquid phase may be considered continuous if any portion of the liquid phase is in fluid contact or communication with any other portion of the liquid. In another example, the liquid phase may be considered continuous if no portion is fully subdivided or divided or otherwise fully physically separated from the rest of the liquid. Optionally, the liquid phase is flowable. Optionally, the continuous liquid phase is not within the gel or matrix. In other embodiments, the continuous liquid phase is within a gel or matrix. For example, the continuous liquid phase occupies pores, interstices or other interstices of the solid or semi-solid support.

One or more primers are optionally immobilized on the support while the liquid phase is within the gel or matrix. Optionally the support is a gel or matrix itself. Alternatively, the support is not a gel or matrix itself. In an example, one primer is immobilized on a solid support contained within a gel and is not immobilized to a gel molecule. The support is for example in the form of a planar surface or one or more microparticles. Optionally the ground plane surface or the plurality of microparticles comprises a forward primer having substantially the same sequence. In embodiments, the support does not comprise a significant amount of a second, different primer. Optionally, the second non-immobilized primer is in solution within the gel. The second non-immobilized primer is, for example, bound to the template strand (i.e., the reverse strand), while the immobilized primer is bound to the forward strand.

Embodiments of template walking include methods of primer extension comprising: (a) a primer-hybridization step, (b) an extension step, and (c) a walking step. Optionally, the primer-hybridization step includes hybridizing a first primer molecule ("first forward primer") to a complementary forward primer binding sequence ("forward PBS") on a nucleic acid strand ("reverse strand"). Optionally, the extending step includes producing an extended first forward strand that is the full-length complement of the reverse strand and hybridizes thereto. The extended first forward strand is generated, for example, by extending the first forward primer molecule in a template-dependent manner using the reverse strand as a template. Optionally, the walking step includes hybridizing a second primer ("second forward primer") to the forward PBS, wherein the reverse strand also hybridizes to the first forward strand. For example, the walking step includes denaturing at least a portion of the forward PBS from the forward strand ("free portion") while another portion of the reverse strand remains hybridized to the forward strand.

In embodiments, the primer extension method is an amplification method comprising template walking, wherein any one or more steps of primer-hybridization, extension, and/or walking are repeated at least once. For example, the method may comprise amplifying the forward strand by one or more amplification cycles. The amplification cycle optionally includes extension and walking. An exemplary amplification cycle comprises or consists essentially of extension and subsequent walking. Optionally, the second forward primer of the first amplification cycle is used as the first forward primer of the subsequent amplification cycle. For example, the second forward primer of the walking step in the first amplification cycle is used as the first forward primer of the extension step of the subsequent amplification cycle.

Optionally, the method of primer extension or amplification further comprises extending or amplifying the reverse strand by: (a) Hybridizing a first reverse primer molecule to a complementary reverse primer binding sequence ("reverse PBS") on the extended forward strand; (b) Generating an extended first reverse strand by extending a first reverse primer molecule in a template-dependent manner using the forward strand as a template, the strand being the full-length complement of the forward strand and hybridizing thereto; and (c) hybridizing a second primer ("second reverse primer") to the reverse PBS, wherein the forward strand is also hybridized to the first reverse strand. Optionally performing one or more repetitions of steps (b) - (c), wherein the second reverse primer of step (c) is the first reverse primer of repeated step (b); and wherein a majority of the forward strand hybridizes to the reverse strand during or all times between the one or more repeats. In embodiments, the majority is optionally at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

Optionally, the reverse strand and/or the forward strand are not exposed to complete denaturing conditions during amplification that result in complete separation of a significant portion (e.g., more than 10%, 20%, 30%, 40%, or 50%) of the multiple strands from their extended and/or full-length complements.

In embodiments, the amplification is performed in one or more amplification cycles (e.g., 1, 5, 10, 20, or allSome amplification cycles) and all times during or between, most of the forward and/or reverse strands optionally hybridize to the extended and/or full-length complements. In embodiments, the majority of the chains are optionally at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the chains. In embodiments, this is accomplished by maintaining the amplification reaction at a higher T than that of the unextended primer _m And lower than T of primer-complementary strand _m Is realized by the temperature of the alloy. For example, the amplification conditions are maintained at a temperature that is higher than the T of the non-extended forward primer _m And less than T of the extended or full length reverse chain _m . Likewise, for example, the amplification conditions are maintained at a temperature that is higher than the T of the unextended reverse primer _m And T below the extended or full length forward chain _m 。

Optionally, one or more of the forward primers and/or one or more of the reverse primers are respirable (e.g., have a low T _m . In examples, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the nucleotide bases of the exhale attractor are adenine, thymine, or uracil or are complementary to adenine, thymine, or uracil.

In some embodiments, the present disclosure generally relates to methods, compositions, systems, devices, and kits for clonally amplifying a nucleic acid template on a support in an amplification reaction solution. Optionally, the nucleic acid template is contacted with the support in a solution comprising a continuous liquid phase. The support may comprise a primer set comprising at least a first primer and a second primer. The primer population may be immobilized on the support, for example, by covalent attachment to the support. In some embodiments, the nucleic acid template comprises a primer binding sequence adjacent to the target sequence. The primer binding sequence may be complementary to the sequence of the first primer and optionally the sequence of the second primer. The target sequence may not be complementary to a primer in the primer set. In some embodiments, the primer binding sequence of the nucleic acid template hybridizes to the first primer. The first primer may be extended along the template using a polymerase, thereby forming an extended first primer. At least a portion of the primer binding sequence of the template may be separated (e.g., denatured or melted) from the extended first primer. Optionally performing separation while maintaining hybridization between the portion of the template and the extended first primer. The portion of the isolated primer binding sequence may then be hybridized to a second primer. Optionally, such hybridization is performed while maintaining hybridization between the other portion of the template and the extended first primer. The second primer can be extended along the template using a polymerase, thereby forming a support comprising the extended first primer and the extended second primer. The extended portion of the extended first primer and/or the extended second primer may comprise a sequence complementary to the target sequence.

In some embodiments, the present disclosure generally relates to a method for clonally amplifying a nucleic acid template on a support in an amplification reaction solution, comprising: contacting a nucleic acid template with a support in a liquid solution, wherein the support comprises an immobilized primer population comprising at least a first primer and a second primer, and wherein the nucleic acid template comprises a primer binding sequence adjacent to a target sequence, wherein the primer binding sequence is complementary to the sequence of the first primer and the sequence of the second primer, and the target sequence is not complementary to a primer in the primer population; hybridizing a primer binding sequence of a nucleic acid template to a first primer; extending the first primer along the template using a polymerase, thereby forming an extended first primer; denaturing at least a portion of the primer binding sequence of the template with the extended first primer while maintaining hybridization between another portion of the template and the extended first primer; hybridizing a portion of the denatured primer binding sequence to the second primer while maintaining hybridization between the other portion of the template and the extended first primer; and extending the second primer along the template using a polymerase, thereby forming a support comprising the extended first primer and the extended second primer, wherein the extension of the extended first primer and the extended second primer each comprise a sequence complementary to the target sequence. The primer population may comprise substantially identical primers differing in sequence by no more than 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the primer population comprises different primers, at least some of which comprise sequences complementary to primer binding sequences of the template. In some embodiments, the primers of the primer population are not complementary to the 5' terminal half sequence of the template. In some embodiments, the primers of the primer population are not complementary to the 3' terminal half sequence of any extended primers of the support. In some embodiments, the primers of the primer population are not complementary to any sequence of the template other than the primer binding sequence.

In some embodiments, the present disclosure generally relates to a method for clonally amplifying a population of nucleic acid templates on a population of supports in an amplification reaction solution, comprising: clonally amplifying a first template on a first support according to any of the methods disclosed herein on a second support according to the same method, wherein all supports are contained within a single continuous liquid phase during amplification.

There is provided, among other things, a method of generating a confined clonal population of immobilized clonal amplicons of a single-stranded template sequence comprising: (a) Ligating a single stranded template sequence ("template 1") to a fixing site ("IS 1"), wherein IS1 comprises multiple copies of an immobilized primer ("IS 1 primer") that can substantially hybridize to template 1, and template 1 binds to IS1 by hybridization to the IS1 primer, and (b) amplifying template 1 in solution using the IS1 primer and a non-immobilized primer ("SP 1 primer"), wherein the amplified strand complementary to single stranded template 1 when single stranded cannot substantially hybridize to the primer on IS1, wherein amplification produces a localized clonal population of immobilized clonal amplicons around the initial hybridization point of template 1 to IS 1.

Also provided IS a method of producing an isolated and immobilized clonal population of a first template sequence ("template 1") and a second template sequence ("template 2"), the method comprising amplifying the first and second template sequences to produce a clonal population of templates 1 that are substantially linked to a first immobilization site ("IS 1") but not a second immobilization site ("IS 2"), or a clonal population of templates 2 that are substantially linked to IS2 but not IS1, wherein: (a) both templates and all amplicons are contained within the same contiguous liquid phase, wherein the contiguous liquid phase IS in contact with first and second immobilization sites (respectively, "IS1" and "IS 2") and wherein IS1 and IS2 are spatially separated, (b) template 1 comprises a first subsequence ("T1-FOR") at one end and a second subsequence ("T1-REV") at its opposite end when in single-stranded form, (c) template 2 comprises a first subsequence ("T2-FOR") at one end and a second subsequence ("T2-REV") at its opposite end, (d) IS1 comprises multiple copies of immobilized nucleic acid primers ("IS 1 primers") that can substantially hybridize to T1-FOR and T2-FOR when T1 and T2 are single-stranded, (e) IS2 comprises multiple copies of immobilized primers ("T2 primers") that can substantially hybridize to T1-FOR and T2 FOR single-stranded FOR use when T1 and T2 are single-stranded FOR use, and (d) IS not substantially complementary to the single-stranded primers when in reverse phase FOR hybridization to the primers; and (g) the reverse complement of T2-REV, when single stranded, does not substantially hybridize to a primer on IS2, but does substantially hybridize to a primer that IS not immobilized in a continuous liquid phase ("SP 2")

Optionally, in any of the methods described herein, any nucleic acid that has dissociated from one of the immobilization sites is capable of substantially hybridizing to the two immobilization sites and any movement of the dissociated nucleic acid to the other immobilization site in the continuous liquid phase (e.g., movement by diffusion, convection) is substantially unimpeded.

Optionally, in any of the methods described herein, the continuous liquid phase IS contacted with IS1 and IS2 simultaneously.

Optionally, in any of the methods described herein, the first portion of the template to which the immobilized primer binds does not overlap with the second portion of the template, the complement of which is bound by the non-immobilized primer.

Optionally, in any of the methods described herein, at least one template to be amplified is generated from the input nucleic acid after contacting the nucleic acid with at least one immobilization site.

Optionally, any of the methods described herein comprise the steps of: (a) Contacting a support comprising immobilized primers with a single stranded nucleic acid template, wherein: hybridizing the first immobilized primer to a Primer Binding Sequence (PBS) on the template; (b) Extending the hybridized first primer with template-dependent extension to form an extended strand that is complementary to the template and at least partially hybridized to the template; (c) Denaturing the template from the extended complementary strand portion so that at least a portion of the PBS is in single stranded form ("free portion"); (d) Hybridizing the free portion to the unextended, immobilized second primer; (e) Extending the second primer with template dependent extension to form an extended strand complementary to the template; (f) Optionally, the annealed extended immobilized nucleic acid strands are separated from each other.

Optionally, in any of the methods described herein, (a) during amplification, a nucleic acid duplex is formed so as to comprise the starting template and/or amplified strand; the duplex is not subjected to conditions that would result in complete denaturation of a substantial number of duplex during amplification.

Optionally, in any of the methods described herein, the single-stranded template is generated by obtaining a plurality of input double-stranded or single-stranded nucleic acid sequences to be amplified (which sequences may be known or unknown) and adding or generating a first universal adaptor sequence and a second universal adaptor sequence on the end of at least one of the input nucleic acids; wherein the first universal adaptor sequence hybridizes to the IS1 primer and/or the IS2 primer and the reverse complement of the second universal adaptor sequence hybridizes to at least one non-immobilized primer. The linker may be double-stranded or single-stranded.

Optionally, in any of the methods described herein, first and second nucleic acid linker sequences are provided at the first and second ends of the single stranded template sequence.

Optionally, in any of the methods described herein, a tag is also added to one or more nucleic acid sequences (e.g., templates or primers or amplicons) that enables identification of the nucleic acid comprising the tag.

Optionally, in any of the methods described herein, all primers on at least one of the immobilization sites or supports have the same sequence. Optionally, the immobilization site or support comprises a plurality of primers having at least two different sequences. In some embodiments, the immobilization site or support comprises at least one target-specific primer.

Optionally, in any of the methods described herein, the continuous medium is flowable. Optionally, the mixing of the non-immobilized nucleic acid molecules is substantially unimpeded in the continuous liquid phase during at least a portion of the amplification process, e.g., during any one or more of the steps or cycles described herein.

Optionally, in any of the methods described herein, mixing is substantially unimpeded during the period of amplification. For example, mixing is substantially unimpeded throughout the duration of amplification.

In embodiments, amplification is achieved using RPA, i.e., recombinase-polymerase amplification (see, e.g., WO2003072805, which is incorporated herein by reference). RPA is optionally performed without substantial change in temperature or reagent conditions. In embodiments herein, partial denaturation and/or amplification can be achieved using a recombinase and/or single-stranded binding protein, including any one or more of the steps or methods described herein. Optionally in combination with a single-chain binding protein (SSB), suitable recombinases include RecA and prokaryotic or eukaryotic analogs thereof or functional fragments or variants thereof. In embodiments, the recombinase agent optionally coats single-stranded DNA (ssDNA), such as amplification primers, to form a nucleoprotein filament strand that invades the template in a double-stranded region of homology. This optionally produces short hybrids and alternative vesicles (displaced strand bubble) (known as D-loops). In embodiments, the free 3' -end of the strand in the D-ring is extended by a DNA polymerase to synthesize a new complementary strand. The complementary strand displaces the originally paired partner strand of the template upon extension. In embodiments, one or more amplification primer pairs are contacted with one or more recombinase agents prior to contact with the optionally double-stranded template.

In any of the methods described herein, amplification of the template (target sequence) comprises contacting a recombinase agent with one or more of at least one amplification primer pair, thereby forming one or more "forward" and/or "reverse" RPA primers. Any recombinase agents not associated with one or more primers are optionally removed. Optionally, one or more forward RPA primers are then contacted with a template strand, optionally having a region complementary to at least one of the RPA primers. The template strand may be hybridized to the contact of the RPA primer with the complementary template, which optionally results in hybridization between the primer and the template. Optionally, the 3' end of the primer is extended along the template using one or more polymerases (e.g., in the presence of dntps) to generate double stranded nucleic acids and displace the template strand. The amplification reaction may include repeated cycles of such contacting and extension until the desired degree of amplification is achieved. Optionally, the displaced strands of the nucleic acid are amplified by a parallel RPA reaction. Optionally, the displaced strand of nucleic acid is amplified by contacting it in turn with one or more complementary primers; and (b) extending the complementary primer by any of the strategies described herein.

In embodiments, the one or more primers comprise a "forward" primer and a "reverse" primer. Contacting both primers with the template optionally results in a first double stranded structure at a first portion of the first strand and a double stranded structure at a second portion of the second strand. Optionally, the 3' ends of the forward and/or reverse primers are extended with one or more polymerases to generate first and second double-stranded nucleic acids and first and second displaced strands of the nucleic acids. Optionally, the second displaced strands are at least partially complementary to each other and can hybridize to form a daughter double stranded nucleic acid, which can be used as a double stranded template nucleic acid in a subsequent amplification cycle.

Optionally the first and the second displaced strand are at least partially complementary to the first or the second primer and are hybridizable to the first or the second primer.

In alternative embodiments of any of the methods or steps or compositions or arrays described herein, the support optionally comprises immobilized primers having more than one sequence. After hybridization of the template nucleic acid strand to the first complementary immobilized primer, the first primer may then be extended and the template and primer may be partially or completely separated from each other. The extended primer may then be annealed to a second immobilized primer having a different sequence than the first primer, and the second primer may be extended. The two extended primers may then be separated (e.g., completely or partially denatured from each other) and may in turn be used as templates for extending additional immobilized primers. The process can be repeated to provide amplified, immobilized nucleic acid molecules. In embodiments, the amplification results in immobilized primer extension products having two different sequences that are complementary to each other, wherein all primer extension products are immobilized to the support at the 5' end.

In some embodiments, the disclosed methods comprise amplification, wherein amplification comprises strand flipping. In the "inverted" embodiment described below, two or more primers are extended to form two or more corresponding extended strands. Optionally, the two or more primers that are extended comprise or consist essentially of substantially the same sequence, and the extended portions of the corresponding extended strands are at least partially different and/or complementary to each other.

One exemplary embodiment of flipping is as follows. The starting template is amplified, for example, by template walking, to generate a plurality of primer-extended strands (which will be referred to as "forward" strands for convenience). Optionally, the forward strand is complementary to the starting template. Optionally, the forward strand is immobilized on a support. Optionally, the forward strands comprise substantially identical sequences, e.g., the forward strands are substantially identical to each other. In embodiments, the forward strand is formed by extending one or more primers immobilized on a support ("forward" primers). The forward primer and/or forward strand is optionally attached to the support at or near its 5' end. Optionally, one or more of the primer-extended forward strands comprises a 3' sequence called a self-hybridizing sequence, which 3' sequence is not present in the unextended primer and can hybridize to a 5' sequence under selected conditions (this process will be referred to as "self-hybridization"). The 5' sequence is optionally part of a forward primer that is not extended. In an example, the forward extension product forms a "stem-loop" structure upon such hybridization. Optionally, the unextended forward primer comprises a "cleavable" nucleotide at or near its 3' end that is susceptible to cleavage. In embodiments, cleavable nucleotides are linked to at least one other nucleotide by "scissile" internucleoside linkages that can be cleaved without substantial cleavage of the phosphodiester linkage.

After extension, the forward-primer extension product (i.e., forward strand) is optionally allowed to self-hybridize. In other embodiments, after allowing self-hybridization, the forward strand is cleaved at the scissile junction of the cleavable nucleotide (e.g., the nucleotide that forms a scissile junction with an adjacent nucleotide). Cleavage results in two fragments of the primer-extension product (i.e., the extended forward strand). In embodiments, the first fragment comprises at least a portion of the original non-extended forward primer. Optionally, the first fragment does not comprise any extended sequences. Optionally, the first fragment is immobilized (e.g., because the non-extended forward primer is already immobilized). In embodiments, the second fragment comprises an extended sequence. Optionally, the second fragment comprises any 3' portion of the unextended primer outside the cleavable nucleotide or does not comprise any portion of the unextended primer. Optionally, the second fragment hybridizes to the first portion by its self-hybridizing sequence.

In an example, a cleavable nucleotide is a nucleotide that is removed by one or more enzymes. The enzyme may for example be a glycosylase. The glycosylase optionally has N-glycosylase activity that releases cleavable nucleotides from double-stranded DNA. Optionally, the removal of the cleavable nucleotide results in an abasic, apurinic or apyrimidinic site. The abasic site may optionally be further modified, for example by another enzymatic activity. Optionally, the abasic site is modified by a lyase to create a base gap. The lyase, for example, cleaves 3 'and/or 5' of the abasic site. Cleavage by a lyase optionally occurs at the 5 'and 3' ends, resulting in removal of abasic sites and leaving base gaps. Exemplary cleavable nucleotides such as 5-hydroxy-uracil, 7, 8-dihydro-8-hydroxyguanine (8-hydroxyguanine), 8-hydroxyadenine, famy-guanine, methyl-famy-guanine, famy-adenine, aflatoxin B1-famy-guanine, 5-hydroxy-cytosine, can be recognized and removed by a variety of glycosylases to form an apurinic site. One suitable enzyme is a carboxamido pyrimidine [ fame ] -DNA glycosylase, also known as 8-hydroxyguanine DNA glycosylase or FPG. FPG can be used as an N-glycosylase and an AP-lyase. The N-glycosylase activity optionally releases the damaged purine from the double stranded DNA, thereby producing a purine-free (AP site), wherein the phosphodiester backbone is optionally intact. The AP-lyase activity cleaves the 3 'and 5' of the AP site to remove the AP site and leave a single base gap. In an example, the cleavable nucleotide is 8-hydroxyadenine, which is converted into a single base gap by FPG having glycosylase and lyase activity.

In another embodiment, the cleavable nucleotide is uridine. Optionally, uridine is cleaved by a "USER" reagent comprising Uracil DNA Glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII, wherein UDG catalyzes cleavage of uracil bases, forming abasic (pyrimidine-free) sites while maintaining the integrity of the phosphodiester backbone, and wherein the lyase activity of the endonuclease VIII breaks the phosphodiester backbone on the 3' and 5' sides of the abasic sites to release abasic deoxyribose, followed by conversion of the phosphate group on the 3' end of the cleavage product to an-OH group, optionally using a kinase.

Optionally contacting at least one of the cleaved fragments with a polymerase. Optionally the first immobilized fragment may be extended by a polymerase. If so desired, the second hybridized fragment can be used as a template for extension of the first fragment. In embodiments, a "flipped" double-stranded extension product is formed. The inverted product may optionally undergo template walking in any of the ways described herein. When both inverted and non-inverted undergo template walking, two distinct extension product populations are formed, wherein the two extension products have identical portions (corresponding to non-extended primers) and portions complementary to each other (corresponding to extended portions of the extension products).

In embodiments, sequences of interest, such as self-hybridizing sequences or new primer binding sites, may optionally be added at the 3' end of the extended forward strand by contacting the extended forward strand with a single stranded "splice" adaptor sequence in the presence of an extension reagent (e.g., a polymerase and dntps). The splice sequence optionally comprises a 3' portion that is substantially complementary to the 3' terminal portion of the extended forward strand and a 5' portion that is substantially complementary to the sequence of interest to be added. After hybridization of the splice junction to the 3' end of the extended forward strand, the forward strand is subjected to template-dependent polymerase extension using the splice junction as a template. Such extension results in the addition of a sequence of interest to the 3' end of the extended forward strand.

Thus, any of the methods of primer extension and/or amplification described herein may comprise any one or more of the following steps: (a) Extending the immobilized forward primer by template walking to generate a plurality of extended forward strands, the forward strands optionally being identical; (b) Optionally hybridizing a splice adaptor to the 3' end of the extended forward strand and subjecting the forward strand to template-dependent polymerase extension using the splice adaptor as a template, thereby adding other 3' sequences to the further extended forward strand, wherein the portion of the added 3' sequence is complementary to the portion of the non-extended forward primer and hybridizes thereto to form a stem-loop structure; (c) Cleaving the forward strand at a frangible junction of cleavable nucleotides at or near the junction of the non-extended forward primer sequence and the extended forward strand sequence; and optionally removing the cleavable nucleotides, thereby producing two cleaved fragments, wherein the first fragment comprises a portion of the non-extended forward primer hybridized to the 3' primer-complementary sequence on the second fragment; (d) Optionally subjecting the first fragment to polymerase extension using the second fragment as a template to generate an inverted forward strand; (e) Optionally hybridizing a second splice junction to the 3' end of the inverted forward strand and subjecting the forward strand to template dependent extension using the splice junction as a template, thereby adding additional 3' sequences to the inverted forward strand, wherein part of the added 3' sequences are new primer binding sequences not present in the inverted strand; (f) The inverted strand comprising the new primer binding sequence is optionally extended or amplified by contact with the new primer and extension or amplification by any method (e.g., as described herein). The new primer will not bind to the inverted strand or the inverted strand that was not further extended in step (e).

FIG. 8 shows a schematic depiction of an exemplary chain flipping and walking strategy. (A) template walking, (B) strand inversion to generate inverted strands, (C) addition of a new primer binding sequence Pg' on the final inverted strand.

Optionally, a single support is used in any of the amplification methods herein, wherein the single support hasThere are multiple primers that can hybridize to the template. In such embodiments, the concentration of the template collector is adjusted prior to contacting the template collector with the solid support such that the individual template molecules in the collector are at least 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ 、4x 10 ⁵ 、5x 10 ⁵ 、6x 10 ⁵ 、8x10 ⁵ 、10 ⁶ 、5x 10 ⁶ Or 10 ⁷ Individual molecules/mm ² Is linked or associated (e.g., by hybridization to a primer immobilized on a solid support).

Optionally, amplifying the individual template molecules in situ on a support, resulting in a population of clones spatially localized around the hybridization point of the starting template. Optionally, the amplification results in no more than about 10 from a single amplified template ² 、10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ ,10 ¹⁰ 、10 ¹¹ 、10 ¹² 、10 ¹⁵ Or 10 ²⁰ Is an amplicon of (a). Optionally, cloning the colonies of amplicons to at least 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ 、4x 10 ⁵ 、5x 10 ⁵ 、6x 10 ⁵ 、8x 10 ⁵ 、10 ⁶ 、5x 10 ⁶ Or 10 ⁷ Individual molecules/mm ² Is located on a solid support.

In some embodiments, the nucleic acid collection may be contacted with one or more supports under conditions wherein multiple nucleic acids are bound to the same support. Such contacting may be particularly useful in methods involving parallel clonal amplification of nucleic acids in different regions of the same support. The ratio of the number of nucleic acids to the surface area of the support can be adjusted to promote monoclonal formation by, for example, ensuring that monoclonal populations of nucleic acids are properly spaced in the support to promote amplified nucleic acids are formed substantially without cross-contamination between different populations of clones. For example, when a single support is used, the collection of nucleic acids to be amplified is adjusted to such dilution that the resulting amplified clonal population produced from individual nucleic acids is generally discontinuous or isolated, e.g., non-overlapping. For example, 50%, 70%, 80%, or 90% or more of the individual nucleic acids within the amplified clonal population are not interspersed with substantially different nucleic acids. Optionally, the different amplification populations are not in contact with or completely overlap with other amplification populations, or can be distinguished from each other using a selected detection method.

In some embodiments, the nucleic acid is attached to the surface of a support. In some embodiments, the nucleic acid may be attached within a support. For example, for a support comprising a hydrogel or other porous matrix, the nucleic acid may be attached to the entire volume of the support, including the surface and within the support.

In some embodiments, a support (or at least one support of a population of supports) may be linked to at least one primer, optionally to a population of primers. For example, the support (or at least one support) may comprise a population of primers. The primers of the primer set may be substantially identical to each other or may comprise substantially identical sequences. One, some, or all of the primers may comprise sequences complementary to sequences within one or more nucleic acid templates. In some embodiments, the primer set may comprise at least two non-complementary primers.

The primer may be attached to the support by its 5 'end and have a free 3' end. The support may be the surface of a slide or the surface of a bead. The primer has a low melting temperature, e.g., oligo (dT) ₂₀ And can hybridize to a low T of a collector linker _m An area. The distance between the primers needs to be shorter than the adaptor length to allow the template to walk, or alternatively, a long primer with a long adaptor at the 5' end will increase the chance of walking.

In some embodiments, the support is ligated and/or contacted with the primer and the template (or reverse strand) under conditions in which the primer and the template hybridize to each other to form a nucleic acid duplex. A double strand may comprise a double-stranded portion comprising the complementary sequences of the template and the primer, wherein at least one nucleotide residue of the complementary sequences are base paired with each other. In some embodiments, the double strand may also comprise a single strand portion. Double strands may also comprise single stranded portions. The single stranded portion may comprise any sequence within the template (or primer) that is not complementary to any other sequence in the primer (or template).

Non-limiting exemplary methods for amplifying nucleic acids cloned on a support are described below. The nucleic acid (which will be referred to as the reverse strand for convenience) is clonally amplified on a support to which are attached multiple copies of complementary forward primers. An exemplary nucleic acid is one of a plurality of DNA collection molecules, e.g., a plurality of nucleic acid members having one or more sequences in common ("adaptors") at their 5 'and/or 3' ends and having variable sequences, e.g., gDNA or cDNA, therebetween. In embodiments, the 3' common moiety, e.g., a linker, has a respirable (e.g., low T _m ) A region, and the 5' common sequence (e.g., linker) optionally has a lower respirable (e.g., higher T _m ) Region, or vice versa. In another embodiment, both the 5 'and 3' common sequences are respirable. Breathable (e.g. low T _m ) The region is, for example, a A, T and/or U-rich region, such as an AT (or U) -rich sequence, such as polyT, polyA, polyU and A, T and U bases or any combination of bases complementary to such bases. Exemplary methods are described herein.

One non-limiting exemplary method of nucleic acid amplification of a clone by "template walking" on a support is shown in FIG. 1. A non-limiting description of an exemplary method of template walking is as follows.

The double stranded DNA library molecules are denatured and the single stranded DNA is attached to the support by primers that hybridize to the support. The ratio of the number of DNA molecules to the support area or the number of beads is set to promote monoclonal formation.

The primer is attached to the primer by its 5 'and has a free 3'. The support may be the surface of a slide or the surface of a bead. The primer has a low melting temperature, e.g., oligo (dT) ₂₀ Or oligo (dA) ₃₀ And can hybridize to low T of library linkers _m An area. The distance between the primers may be shorter than the adaptor length to allow the template to walk, or alternatively, a long primer with a long adaptor at the 5' end will increase the chance of walking.

Clonally amplifying nucleic acids on a support to which a plurality ofAnd (3) copying the primer. An exemplary nucleic acid is one of a plurality of DNA library molecules, e.g., having one or more sequences in common (e.g., "linkers") at their 5 'and/or 3' ends and having variable sequences therebetween, e.g., gDNA or cDNA. In embodiments, the 3' linker has a low T _m Region, and 5' linker optionally has a higher T _m Region, or vice versa. Low T _m The region is, for example, a pyrimidine-rich region, such as an AT (or U) -rich sequence, such as polyT, polyA, polyU and A, T and U bases or any combination of bases complementary to such bases. Exemplary methods are described herein.

The one or more primers, whether in dissolved form or attached to a support, are incubated with a DNA polymerization or extension reaction mixture, optionally comprising any one or more reagents such as enzymes, dntps, and buffers. Extension primers (e.g., forward primers). Optionally, extension is template-dependent extension of the primer along the template, including the sequential incorporation of nucleotides that are each complementary to a sequential nucleotide on the template, such that an extended or unextended forward primer is complementary to the reverse strand (also referred to as antiparallel or complementary). Optionally, the extension is achieved by an enzyme having polymerase activity or other extension activity, such as a polymerase. The enzyme may optionally have other activities including 3'-5' exonuclease activity (proofreading activity) and/or 5'-3' exonuclease activity. Alternatively, in some embodiments, the enzyme may lack one or more of these activities. In embodiments, the polymerase has strand displacement activity. Examples of useful strand displacement polymerases include bacteriophage Φ29DNA polymerase and Bst DNA polymerase. Optionally, the enzyme is active at elevated temperatures, e.g., at or above 45 ℃, at 50 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ or 85 ℃.

An exemplary polymerase is Bst DNA polymerase (exonuclease negative), which is a 67kDa bacillus stearothermophilus (Bacillus stearothermophilus) DNA polymerase protein (large fragment) (exemplified by accession number 2bdp_a) that has 5'-3' polymerase activity and strand displacement activity that lacks 3'-5' exonuclease activity alone. Other polymerases include Taq DNA polymerase I (exemplified by accession number 1 TAQ) from Thermus aquaticus (Thermus aquaticus), eco DNA polymerase I (accession number P00582) from Escherichia coli, aea DNA polymerase I (accession number 067779) from Thermus hyperthermophilus (Aquifex aeolicus), or functional fragments or variants thereof, e.g., functional fragments or variants having at least 80%, 85%, 90%, 95% or 99% sequence identity at the nucleotide level.

Typically, the extension step produces a nucleic acid comprising a double-stranded duplex portion in which two complementary strands hybridize to each other. In one embodiment, walking comprises subjecting the nucleic acid to partial denaturing conditions that denature a portion of the nucleic acid strand but not enough to completely denature the nucleic acid over its entire length. In embodiments, the nucleic acid is not subjected to complete denaturation conditions during part or all of the duration of the walking procedure.

In embodiments, the sequences of the negative and/or positive strands are designed such that the primer binding sequence or portion thereof is breathable, i.e., susceptible to denaturation under selected conditions (e.g., amplification conditions). The respirable fraction is optionally more susceptible than a majority of nucleic acids of similar length having a random sequence, or than at least another fraction of the strand comprising the respirable sequence. Optionally, the respirable sequence exhibits a significant amount of denaturation under the amplification conditions selected (e.g., at least 10%, 20%, 30%, 50%, 70%, 80%, 90% or 95% of the molecules are fully denatured over the respirable sequence). For example, the respirable sequence is designed to be completely denatured in 50% of the chain molecules at 30, 35, 40, 42, 45, 50, 55, 60, 65 or 70 ℃ under selected conditions (e.g., amplification conditions).

When partial denaturation is achieved by heating or elevated temperatures, exemplary respirable PBSs may be pyrimidine-rich (e.g., have high levels of a and/or T and/or U). PBS contains, for example, poly-A, poly-T or poly-U sequences or bundles of polypyrimidines. One or more amplification or other primers (e.g., immobilized primers) are optionally designed to be correspondingly complementary to these primer binding sequences. Exemplary PBS for the nucleic acid strand comprises a poly-T sequence, e.g., a segment of at least 10, 15, 20, 25, or 30 thymidine nucleotides While the corresponding primer has a complementary sequence to PBS, e.g., a segment of at least 10, 15, 20, 25 or 30 adenosine nucleotides. Exemplary low melting primers optionally have such a high proportion (e.g., at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of nucleobases: which typically form no more than two hydrogen bonds with complementary bases when the primer hybridizes to the complementary template (e.g., under selected amplification conditions). Examples of such nucleobases include a (adenine), T (thymine) and U (uracil). Exemplary low melting primers optionally have a high proportion of any one or more of a (adenine), T (thymine), and/or U (uracil) or derivatives thereof. In embodiments, the derivative comprises nucleobases complementary to a (adenine), T (thymine), and/or U (uracil). The portion of the primer that hybridizes to PBS optionally has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% a (adenine), T (thymine) or U (uracil) nucleotides, or any combination thereof. In another example, the portion of the primer that hybridizes to PBS comprises a polyA sequence (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides in length). Other exemplary primers include (NA _x ) _n A repeating sequence. Optionally, n (lower case) is 2-30, such as 3-10, such as 4-8."N" (in uppercase) is any nucleotide-and optionally N is C or G. "A" is an abbreviation for adenine and "x" indicates the number of adenine residues in the repeat sequence, e.g., 2, 3, 4, 5, 6, 10 or more. Exemplary primer Contains (CAA) _n 、CA) _n 、(CAAA) _n Or even (GAA) _n Is a sequence of multiple repeats of (a).

Optionally only one strand (e.g., the forward or reverse strand) has breathable PBS. In another embodiment, both the forward and reverse chains have breathable PBS. Breathable PBS is optionally complementary to immobilized to a support or non-immobilized (e.g., solubilized) primer. Optionally, the chains comprising breathable PBS are immobilized to a support or are not immobilized (e.g., in dissolved form). Optionally both primers are immobilized, or both strands are immobilized. Optionally none of the primers is immobilized, or none of the strands is immobilized.

The amplification cycle optionally includes respiration (breathing), annealing, and extension. Optionally subjecting the nucleic acid to be amplified to conditions suitable or optimal for at least one of these steps. In embodiments, the nucleic acid is subjected to conditions suitable for more than one of these steps (e.g., annealing and extension, or respiration and extension). In some examples, all three of these steps may occur simultaneously under the same conditions.

In an exemplary method, the nucleic acid may be subjected to conditions that allow or promote respiration. In embodiments, a "breath" is considered to occur when the two strands of a double-stranded duplex are substantially hybridized to each other but denatured in the local portion of interest. The first complementary strand (e.g., the forward or reverse strand) to which one or more respirable sequences of a nucleic acid (e.g., forward and/or reverse PBS having a low Tm portion) hybridize is locally denatured ("respired") and thus available for hybridization to another second strand. An exemplary first strand is a primer extension product from a first primer. Exemplary second strands are, for example, second unextended primers (e.g., PBS complementary oligonucleotides comprising, for example, dT or dA sequences). Optionally, the first and second chains are immobilized on a support, and may be closely positioned (e.g., sufficiently closely adjacent to allow walking). The conditions used for respiration are optionally partially denaturing conditions under which PBS is substantially denatured but another portion of the nucleic acid remains hybridized or double stranded. Optionally, a DNA helicase may be included in the reaction mixture to promote partial denaturation.

Optionally, the nucleic acid is then subjected to conditions that promote annealing, e.g., reducing the temperature, to enable hybridization between the breathable PBS and the second strand. In embodiments, the same conditions are used to promote respiration and elongation. In another embodiment, the annealing conditions are different from the respiration conditions-e.g., the annealing conditions are non-denaturing conditions or conditions that promote denaturation less than the respiration conditions. In an example, the annealing conditions include a lower temperature (e.g., 37 ℃) than the breathing conditions in which a higher temperature (e.g., 60-65 ℃) is used. Optionally, complete denaturation conditions are avoided during one or more amplification cycles (e.g., most amplification cycles or substantially all amplification cycles).

Optionally, one or more PBS-respiration and primer extension steps are repeated multiple times to amplify the starting nucleic acid. When one or more nucleic acid reagents (e.g., primers) are immobilized to a support, the primer-extension product substantially remains attached to the support, e.g., due to attachment of an extended primer to the support that was not extended prior to amplification or by hybridization to such primer.

Optionally, preparing a sample of one or more populations of nucleic acids to be amplified. The population of nucleic acids may be in single-stranded or double-stranded form; optionally the one or more nucleic acids each comprise a nucleic acid strand having a known 3 'terminal sequence and a known 5' terminal sequence that is substantially identical or complementary to the one or more primers used for amplification. The 3 'portion of the nucleic acid strand may be, for example, complementary to the immobilized primer, while the 5' portion may be identical to the solubilized primer. The 5 'and/or 3' portions may be common ("universal") or unchanged between individual nucleic acids within a population. Optionally, the nucleic acids within the population each comprise a different (e.g., unknown) sequence between the common portions, e.g., genome DNA, cDNA, mRNA, mate-pair (mate-pair) fragments, exomes, etc. The collection may, for example, have sufficient members to ensure coverage of greater than 50%, 70% or 90% of the corresponding genetic source.

In some embodiments, the present disclosure relates generally to compositions for nucleic acid amplification comprising reaction mixtures for nucleic acid amplification and related systems, devices, kits, and methods.

In some embodiments, the present disclosure generally relates to compositions for nucleic acid amplification comprising a reaction mixture comprising a continuous liquid phase comprising (i) a polymerase and (ii) a plurality of supports, at least one of which is linked to a substantially monoclonal nucleic acid population, and related systems, devices, kits, and methods.

In some embodiments, the present disclosure relates generally to compositions for nucleic acid amplification comprising a reaction mixture comprising a continuous liquid phase comprising (i) a polymerase and (ii) a plurality of supports including a first support and a second support, and related systems, devices, kits, and methods.

In some embodiments, the present disclosure relates generally to compositions (and related systems, devices, kits, and methods) for nucleic acid amplification comprising: a reaction mixture comprising a continuous liquid phase comprising (i) a plurality of supports including a first support and a second support, (ii) a plurality of different polynucleotides comprising a first polynucleotide and a second polynucleotide, and (iii) reagents for isothermal nucleic acid amplification. In some embodiments, the reagents for nucleic acid amplification comprise a polymerase and one or more types of nucleotides (e.g., a plurality of nucleotides). Optionally, the reagent for isothermal nucleic acid amplification comprises a recombinase.

Optionally, the first and second polynucleotides have different sequences.

Optionally, at least one end of at least one of the plurality of different polynucleotides is linked to at least one oligonucleotide adaptor.

Optionally, at least one end of at least some of the plurality of different polynucleotides comprises a common sequence.

Optionally, at least two of the different polynucleotides in the reaction mixture comprise a common sequence.

Optionally, the first and second polynucleotides are different.

In some embodiments, the liquid phase comprises one or more supports of the plurality comprising primers.

Optionally, the reaction mixture comprises a continuous liquid phase.

Optionally, the reaction mixture may be used to perform isothermal or thermocycling nucleic acid amplification.

Optionally, the continuous liquid phase comprises any one or any combination of (i) one or more polymerases and/or (ii) at least one support.

Optionally, the continuous liquid phase comprises a plurality of supports.

Optionally, the continuous liquid phase comprises a first support.

Optionally, the continuous liquid phase comprises a second support.

Optionally, at least one support of the plurality may be linked to a substantially monoclonal nucleic acid population.

Optionally, the first support may be linked to a first substantially monoclonal nucleic acid population.

Optionally, a second support may be attached to a second substantially monoclonal nucleic acid population.

Optionally, the first and second substantially monoclonal nucleic acid populations comprise different sequences or substantially identical sequences.

Optionally, the first and second substantially monoclonal nucleic acid populations hybridize or do not hybridize to each other under stringent hybridization conditions.

Optionally, the first and second substantially monoclonal nucleic acid populations are not identical.

Optionally, the first and second substantially monoclonal nucleic acid populations are non-complementary.

Optionally, the reaction mixture comprises nucleotides that are not exogenously labeled. For example, the nucleotide may be a naturally occurring nucleotide, or a synthetic analog that does not contain a fluorescent moiety, dye, or other exogenous optically detectable label.

Optionally, the reaction mixture is contained in a single reaction vessel.

Optionally, the reaction mixture comprises an isothermal or thermocycled reaction mixture.

Optionally, the plurality of supports comprises beads, particles, microparticles, spheres, gels, filters, or the inner wall of a tube.

Optionally, at least one support of the plurality may be linked to a plurality of nucleic acids.

Optionally, at least one support of the plurality may be attached to one or more primers. The primers may be identical (or comprise a common sequence) or different.

Optionally, at least one support may be attached to the plurality of first primers.

Optionally, at least one support may be attached to the plurality of first primers and the plurality of second primers.

Optionally, the plurality of first primers comprises substantially the same sequence.

Optionally, the plurality of first primers comprises at least one first primer comprising a sequence that is identical or complementary to at least a portion of a polynucleotide of the plurality of different polynucleotides.

Optionally, the plurality of second primers comprises at least one second primer comprising a sequence that is identical or complementary to at least a portion of the polynucleotides of the plurality of different polynucleotides.

In some embodiments, at least one polynucleotide of the plurality of different polynucleotides comprises a first sequence that is substantially identical or substantially complementary to a sequence within the first primer. In some embodiments, the at least one polynucleotide further comprises a second sequence that is substantially identical or substantially complementary to a sequence within the second primer. In some embodiments, substantially all of the polynucleotides in the plurality of different polynucleotides comprise a first sequence and a second sequence.

Optionally, at least one support of the plurality is linked to 2-10 different plurality of primers.

Optionally, 2-10 different pluralities of primers comprise different sequences.

Optionally, 2-10 different plurality of primers comprise at least one sequence that hybridizes to at least a portion of a different polynucleotide.

Optionally, 2-10 different plurality of primers comprise at least one sequence that hybridizes to at least a portion of a common sequence in different polynucleotides.

Optionally, at least one of the supports is linked to at least one uniquely identifying barcode sequence.

Optionally, the first and second substantially monoclonal nucleic acid populations have substantially the same or different sequences.

Optionally, the reaction mixture comprises at least one recombinase.

Optionally, the recombinase may catalyze homologous recombination, strand invasion and/or D-loop formation.

Optionally, the recombinase is part of a nucleoprotein filament comprising the recombinase linked to a primer attached to a support in a reaction mixture. The primer linked by the recombinase may be attached to the support or in solution.

Optionally, the reaction mixture comprises a nucleoprotein complex or a plurality of nucleoprotein complexes.

Optionally, the reaction mixture comprises a first nucleoprotein complex.

Optionally, the reaction mixture comprises a second nucleoprotein complex.

Optionally, at least one nucleoprotein complex of the plurality comprises at least one recombinase linked to a primer.

Optionally, the reaction mixture comprises a first nucleoprotein complex containing at least one recombinase enzyme linked to a first primer.

Optionally, the reaction mixture comprises a second nucleoprotein complex containing at least one recombinase enzyme linked to a second primer.

Optionally, the recombinase comprises a phage recombinase from T4, T2, T6, rb69, aeh, KVP40, acinetobacter (Acinetobacter) phage 133, aeromonas (Aeromonas) phage 65, blue-green algae phage P-SSM2, blue-green algae phage PSSM4, blue-green algae phage S-PM2, rb14, rb32, aeromonas phage 25, vibrio (Vibrio) phage nt-1, phi-1, rb16, rb43, phage 31, phage 44RR2.8t, rb49, phage Rb3, or phage LZ 2.

Optionally, the recombinase comprises uvsX recombinase from a T4 bacteriophage or recA recombinase from E.coli.

Optionally, the reaction mixture further comprises a polymerase.

Optionally, the polymerase lacks 5 'to 3' exonuclease activity.

Optionally, the polymerase comprises strand displacement activity.

Optionally, the polymerase comprises a thermostable or thermosensitive polymerase.

Optionally, the polymerase comprises a DNA polymerase or an RNA polymerase.

Optionally, the reaction mixture further comprises at least one type of nucleotide.

Optionally, at least one support of the plurality comprises at least one primer.

Optionally, the first support is linked to a first substantially monoclonal nucleic acid population and the second support is linked to a second substantially monoclonal nucleic acid population.

Optionally, the first and second substantially monoclonal nucleic acid populations have different nucleic acid sequences.

Optionally, the first and second substantially monoclonal nucleic acid populations do not hybridize to each other under stringent hybridization conditions.

Optionally, the first and second nucleic acids are substantially monoclonal nucleic acid populations that are non-identical and non-complementary.

Optionally, the reaction mixture comprises (i) at least two polynucleotide templates to be amplified and/or (ii) at least one nucleoprotein filament complex.

Optionally, the reaction mixture comprises at least one polynucleotide, primer, template, or amplification product linked to a drag compound. As used herein, the term "drag compound" and variants thereof describe any chemical composition that: the composition can attach to a nucleic acid and hinder its diffusion through the reaction mixture, but still allow for nucleic acid synthesis using such polynucleotides, primers, templates, or amplification products in a nucleic acid synthesis reaction. The attachment of such drag compounds to nucleic acids within a synthesis reaction generally reduces the mobility of such nucleic acids in the reaction mixture and can be used to prevent cross-contamination of amplified products or templates between different synthesis reactions occurring using the same reaction mixture. In some embodiments, the attachment of the drag component to the one or more nucleic acid components may increase the number or proportion of monoclonal products.

In some embodiments, the present teachings provide methods for nucleic acid amplification comprising at least one fluidity-altering nucleic acid (e.g., primer). In some embodiments, the fluidity-altered nucleic acid exhibits increased or decreased fluidity through an aqueous medium. In some embodiments, the modified nucleic acid comprises a nucleic acid (e.g., a primer) linked at any position along the length of the nucleic acid to one or more compounds that alter the mobility of the nucleic acid through an aqueous medium (e.g., resistance compounds). In some embodiments, a resistance compound that alters the fluidity of a nucleic acid can be attached to any primer in a nucleic acid amplification reaction, including a first, second, third, fourth, or any other primer. For example, one or more drag compounds can be attached to a nucleic acid at any one or any combination of the 5 'terminus, the 3' terminus, and/or the internal position. In some embodiments, the modified nucleic acid may be covalently or non-covalently linked to a resistance compound that alters the mobility of the nucleic acid through the aqueous medium. For example, the drag compound, when attached to a nucleic acid, can provide hydrodynamic drag of water by changing the physical size, length, radius, shape, or charge of the modified nucleic acid as compared to the nucleic acid lacking the attached compound. In some embodiments, the drag compound attached to the nucleic acid can alter the interaction between the nucleic acid and the aqueous medium (as compared to the interaction between the aqueous medium and the nucleic acid lacking the attached compound). In some embodiments, the drag compound may be synthetic, recombinant, or naturally occurring. In some embodiments, the drag compound may be dotted, uncharged, polar or hydrophobic. In some embodiments, the resistance compound may be linear, branched, or have a dendritic polymeric structure (dendrimeric structure). In some embodiments, the drag compound may comprise a single moiety or polymer of nucleosides, sugars, lipids, or amino acids.

Optionally, the drag compound comprises a sugar moiety, polysaccharide, protein, glycoprotein, or polypeptide. Optionally, the drag compound comprises BSA, lysozyme, β -actin, myosin, whey protein, ovalbumin, β -galactosidase, lactate dehydrogenase, or an immunoglobulin (e.g., igG).

Optionally, the resistance compound that alters the fluidity of the nucleic acid through the aqueous medium comprises one or more polyethylene oxide (PEO) or polypropylene oxide (PPO) moieties, including polymers of polyethylene oxide (PEO) or polypropylene oxide (PPO). Non-limiting examples of such polymers include triblock copolymers (e.g., PEO-PPO-PEO), pluronics ^TM -a polymer of the type and a hydrophobically modified PEO polymer. Optionally, the drag compound comprises one or more amino acid moieties, polypeptides, and clustered peptides. Optionally, the drag compound includes a sugar moiety, a polysaccharide, a hydrophobically modified polysaccharide, a cellulose derivative, sodium carboxymethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, or hydroxypropyl methyl cellulose. Optionally, the resistance compound comprises a hydrophobically modified alkali soluble associative (HASE) polymer, a hydrophobically modified polyacrylamide, a heat sensitive polymer, or N-isopropylacrylamide (NTPAAm), optionally the resistance compound comprises poly (ethylene glycol) methyl ether acrylate (PEGMEA), tetraethylene glycol diacrylate (TEGDA), poly (ethylene glycol) dimethacrylate (EGDMA), or N, N' -methylene-bis-acrylamide (NMBA).

Optionally, the drag compound comprises a protein or polypeptide including BSA, lysozyme, β -actin, myosin, whey protein, ovalbumin, β -galactosidase, or lactate dehydrogenase. In some embodiments, the drag compound may be attached to the nucleic acid by an amine or sulfhydryl linkage.

In some embodiments, the fluidity-altering nucleic acid comprises a nucleic acid linked to a binding partner (e.g., an affinity moiety for interacting with a receptor moiety). In some embodiments, the acceptor moiety may be used as a drag compound. In some embodiments, an affinity moiety may be attached to the nucleic acid, and the affinity moiety (which serves as a drag compound) interacts with the receptor moiety. For example, the nucleic acid may be linked to a biotin moiety that can bind to the avidin-like moiety. The avidin-like moiety may be used as a drag compound. Avidin-like moieties include avidin, any derivatives, analogs, and other non-natural forms of avidin that can bind to a biotin moiety. Other examples of binding partners include epitopes (e.g., protein a) and their respective antibodies (e.g., anti-FLAG antibodies), as well as fluorescein and anti-fluorescein antibodies. One skilled in the art will readily recognize other combinations of binding partners for attaching a drag compound to a nucleic acid.

Optionally, the drag compound can be attached to the primer by attaching the drag compound and the primer to each of the two members of the binding partner pair.

Optionally, at least one primer in the reaction mixture comprises biotin.

Optionally, the resistance compound comprises avidin or streptavidin.

Optionally, the drag compound comprises a sugar moiety, polysaccharide, protein, glycoprotein, or polypeptide.

Optionally, the drag compound comprises BSA, lysozyme, β -actin, myosin, whey protein, ovalbumin, β -galactosidase, lactate dehydrogenase, or an immunoglobulin (e.g., igG).

Optionally, the reaction mixture further comprises an accessory protein.

Optionally, the accessory protein comprises a helicase, a single chain binding protein, or a recombinase loading factor.

Optionally, the helicase comprises uvsW from a T4 phage.

Optionally, the single chain binding protein comprises an Sso SSB from sulfolobus solfataricus (Sulfolobus solfataricus), an MjA SSB from Methanococcus jannaschii (Methanococcus jannaschii), or an E.coli SSB protein.

Optionally, the single-chain binding protein comprises gp32 protein from a T4 bacteriophage or a modified gp32 protein from a T4 bacteriophage.

Optionally, the recombinase-loaded protein comprises uvsY from a T4 bacteriophage.

Optionally, the reaction mixture further comprises ATP.

Optionally, the reaction mixture further comprises an ATP regeneration system.

Optionally, the ATP regeneration system comprises creatine phosphate.

Optionally, the ATP regeneration system comprises creatine kinase.

Optionally, the reaction mixture further comprises adducts for increasing the efficiency or yield of the nucleic acid amplification reaction.

Optionally, the adducts include betaine, DMSO, proline, trehalose, MMNO (4-methylmorpholine N-oxide) or PEG-like compounds.

Optionally, at least one polynucleotide template in the reaction mixture comprises a first sequence that is complementary to or identical to at least some portion of the first primer, and a second sequence that is complementary to or identical to at least some portion of the second primer. Optionally, the reaction mixture comprises a plurality of double stranded polynucleotides comprising a first sequence complementary to or identical to at least some portion of the first primer and a second sequence complementary to or identical to at least some portion of the second primer. Optionally, the first sequence is located at or near the end of at least one double stranded polynucleotide of the plurality and the second sequence is located at or near the other end of at least one double stranded polynucleotide of the plurality.

Optionally, the reaction mixture further comprises a diffusion limiting agent.

Optionally, the diffusion limiting agent reduces the rate of diffusion of the polynucleotide away from the support.

Optionally, the diffusion limiting agent reduces the level of the polyclonal nucleic acid population attached to the support.

Optionally, the diffusion limiting agent comprises a polymer compound.

Optionally, the diffusion limiter comprises a sugar polymer.

Optionally, the diffusion limiter comprises a cellulose-based compound.

Optionally, the diffusion limiting agent comprises a glucose or galactose polymer.

Optionally, the sugar polymer comprises cellulose, dextran, starch, glycogen, agar or agarose.

Optionally, the diffusion limiting agent comprises a block copolymer compound.

Optionally, the diffusion limiter comprises a poly (propylethylene oxide) central chain flanked by two poly (ethylene oxide) hydrophilic chains.

Optionally, the diffusion limiter forms micelles.

Optionally, the diffusion limiter forms a micellar liquid crystal.

Optionally, the diffusion limiting agent comprises Pluronics ^TM A compound.

Optionally, the reaction mixture further comprises a diffusion reducing agent at a concentration of about 0.025-0.8% w/v, or about 0.05-0.7% w/v, or about 0.075-0.6% w/v, or about 0.1-0.5% w/v, or about 0.2-0.4% w/v.

Optionally, the compositions for nucleic acid amplification and related systems, devices, kits, and methods further comprise a surface, matrix, or medium comprising a plurality of sites, wherein at least one site is operably coupled to one or more sensors.

Optionally, the plurality of sites includes a reaction chamber, a support, particles, microparticles, spheres, beads, a filter, a flow cell, a well, a trench, a groove receptacle, an inner wall of a gel or a tube.

Optionally, the plurality of sites may be arranged in a random array or a coded array.

Optionally, multiple sites may be in fluid communication with each other.

Optionally, at least one of the plurality of sites comprises a three-dimensional chemistry matrix.

Optionally, at least one of the plurality of sites may be covalently linked to the three-dimensional chemistry matrix.

Optionally, at least one of the plurality of sites comprises an acrylamide layer. Optionally, at least one of the plurality of sites comprises a nucleic acid covalently linked to an acrylamide layer.

Optionally, the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

Optionally, the hydrophilic polymer matrix is an in situ cured polymer matrix.

Optionally, polyacrylamide is conjugated to the oligonucleotide primer.

Optionally, the holes have a depth of 0.01 microns to 10 microns.

In some embodiments, the sensor includes a Field Effect Transistor (FET). The FETs may include Ion Sensitive FETs (ISFETs), chemosensitive field effect transistors (chemfets), or bioactive field effect transistors (biofets).

Optionally, one or more sensors are configured to detect byproducts of nucleotide incorporation.

Optionally, one or more sensors may be configured to detect the presence of chemical moieties at one or more of the plurality of sites.

Optionally, the one or more sensors include a Field Effect Transistor (FET), an Ion Sensitive Field Effect Transistor (ISFET), a chemosensitive field effect transistor (chemFET), or a bioactive field effect transistor (bioFET).

In some embodiments, the sensing material comprises a metal-oxide.

In some embodiments, the sensing material is sensitive to hydrogen ions.

Optionally, byproducts from the nucleotide incorporation reaction include pyrophosphates, hydrogen ions, or protons.

Also provided are compositions comprising any, any subgroup or all of the following: at least one reverse nucleic acid strand, a plurality of forward primers immobilized on at least one support, a plurality of reverse primers in solution, and a polymerase. The forward and/or reverse primers are optionally low melting or rich in adenine, thymine or uracil as described herein. Exemplary compositions include a solid support comprising a plurality of spatially separated clonal populations each comprising a low melting point primer binding sequence at the 3 'end and a low melting point primer sequence at the 5' end. Optionally, the composition further comprises a recombinase enzyme. Alternatively, the composition optionally does not comprise another enzyme that is not a polymerase, such as a recombinase or reverse transcriptase or helicase or nicking enzyme. Another exemplary composition comprises any one or more of the following components: (1) a reverse nucleic acid strand, (2) a plurality of low melting forward primers immobilized on a support, (3) a plurality of low melting reverse primers in solution, and (4) a polymerase. Optionally, the forward primer is hybridizable (e.g., complementary) to the 3' portion or end of the reverse strand. Optionally, the reverse primer is substantially identical to the 5' portion or end of the reverse strand. The composition may comprise any one or more of the agents described herein, and/or may undergo any one or more of the procedures or conditions described herein.

In some embodiments, the disclosure generally relates to compositions comprising amplified nucleic acids produced by any of the methods of the disclosure. In some embodiments, a localized clonal population of clonal amplicons is formed around discrete sites on a support. An exemplary discrete site is the point of attachment of the starting nucleic acid strand to the support, and from this site other nucleic acids within the clonal population are directly or indirectly generated by primer extension using the starting nucleic acid or a copy thereof as a template.

Optionally, the composition comprises a collection of nucleic acids that can be produced by any one or more of the methods described herein. For example, the collection can include immobilized nucleic acids that occupy one or more separate regions on the surface. In some embodiments, each region comprises a plurality of identical nucleic acid strands and optionally a plurality of identical complementary strands hybridized thereto, wherein the complementary strands are not attached to or linked to or associated with the solid support except for those resulting from hybridization to the immobilized nucleic acid. Optionally, the individual nucleic acid strands within such regions are positioned such that the other nucleic acid strand is located on the surface within a distance of the length of the strand. Per mm of surface optionally having nucleic acid immobilized thereon ² There is at least one discrete area. For example, the number of separated regions/mm ² A surface having nucleic acid immobilized thereon of more than 10 ² Is greater than 10 ³ Is greater than 10 ⁴ Is greater than 10 ⁵ Is greater than 10 ⁶ Is greater than 10 ⁷ Or greater than 10 ⁸ 。

The collection of amplified clonal populations can form an array, which can be one-dimensional (e.g., a column of generally monoclonal microbeads) or two-dimensional (e.g., the amplified clonal populations reside on a planar support) or three-dimensional. The individual clonal populations of the array are optionally, but not necessarily, positioned or arranged so that they are addressed or addressable. Optionally, the different clonal populations are spaced from each other by a suitable distance that is generally sufficient to allow the different clonal populations to be distinguished from each other. In embodiments, the localized clonal population is spread over a planar substrate in an ordered or unordered (e.g., random) manner.

An exemplary array is characterized by an individual distinguishable population of nucleic acid clones, wherein the features are optionally distributed on one or more supports. In exemplary bead embodiments, the array comprises a plurality of beads, wherein individual beads typically comprise a monoclonal population of nucleic acids, and different beads typically comprise different clonal populations (e.g., which differ in sequence). Optionally, the microbeads are distributed or packed in a monolayer on a planar substrate. In other embodiments, the array comprises a single (e.g., planar) support comprising a plurality of spatially discontinuous populations of nucleic acid clones, wherein the different populations of clones optionally differ in sequence.

Optionally, one or more nucleic acids within an individual clonal population can be directly linked to a planar substrate. In another example, the nucleic acids of the individual clonal populations are linked to microbeads, e.g., as discussed herein. The cloned microbeads are optionally packed tightly together in a random or ordered manner on a planar substrate. Optionally, more than 20%, 30%, 50%, 70%, 80%, 90%, 95% or 99% of the microbeads are contacted with at least one, two, four or six other microbeads. Optionally, less than 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or 99% of the microbeads are contacted with one, two, four or six other microbeads.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification and related compositions, systems, kits, and devices, including multiplex nucleic acid amplification using any of the amplification methods, compositions, or systems disclosed herein.

In some embodiments, the method comprises performing multiplex amplification (e.g., polymerase-mediated multiplex nucleic acid amplification reactions) using a polymerase.

In some embodiments, the method may further comprise re-amplifying the amplicon from the multiplex nucleic acid amplification using a nucleic acid amplification reaction.

Optionally, multiplex nucleic acid amplification can be performed in a single reaction mixture.

Optionally, a sample comprising a plurality of different nucleic acid target sequences may be subjected to multiplex nucleic acid amplification.

Optionally, a plurality of different nucleic acid target sequences can be amplified in a single reaction mixture.

Optionally, at least a few dozen or at least hundreds or at least thousands (or more) of nucleic acid target sequences may be amplified in a single reaction mixture.

Optionally, at least fifty or at least one hundred nucleic acid target sequences can be amplified in a single reaction mixture.

Optionally, multiplex amplification may comprise contacting at least a portion of the sample with any one or any combination of a recombinase, a polymerase, and/or at least one primer.

Optionally, multiplex amplification may be performed under isothermal or thermocycling conditions.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification, and related compositions, systems, kits, and devices, including multiplex nucleic acid amplification comprising amplifying at least fifty (or more) different nucleic acid target sequences from a sample comprising a plurality of different nucleic acid target sequences within a single reaction mixture, the amplifying comprising contacting at least a portion of the sample with a recombinase and a plurality of primers under isothermal amplification conditions.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification, and related compositions, systems, kits, and devices, including multiplex nucleic acid amplification comprising amplifying different nucleic acid target sequences from a sample comprising the plurality of different nucleic acid target sequences within a single reaction mixture, the amplification comprising generating a plurality of at least fifty different amplified target sequences (or more) by contacting at least a portion of the sample with a recombinase and a plurality of primers under isothermal amplification conditions.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification, and related compositions, systems, kits, and devices, including generating a substantially monoclonal nucleic acid population by re-amplifying amplicons from multiple nucleic acid amplifications using a nucleic acid amplification reaction (e.g., a recombinase).

Optionally, the method for multiplex nucleic acid amplification may further comprise a recombinase-mediated nucleic acid amplification method comprising re-amplifying at least some of the at least fifty different amplified target sequences by: (a) Forming a reaction mixture comprising a single continuous liquid phase comprising (i) a plurality of supports, (ii) at least one of fifty different amplified target sequences, and (iii) a recombinase; and (b) subjecting the reaction mixture to amplification conditions, thereby producing a plurality of supports linked to a substantially monoclonal population of nucleic acids linked thereto.

Optionally, in a method for multiplex nucleic acid amplification, different nucleic acid target sequences from a sample may be amplified under substantially non-depleting conditions.

Optionally, in a method for multiplex nucleic acid amplification, different nucleic acid target sequences from a sample may be amplified under substantially depleted conditions.

Optionally, in the method for multiplex nucleic acid amplification, the single reaction mixture comprises an isothermal or thermocycling reaction mixture.

In some embodiments, two or more templates or targets may be amplified within separate chambers, wells, cavities, or sites of an array that are in fluid communication with each other or occupied by the same single continuous liquid phase of the amplification reaction mixture. Such embodiments include embodiments for array-based nucleic acid amplification.

For example, in some embodiments, the present disclosure relates to methods for nucleic acid amplification, comprising: partitioning the target polynucleotides into reaction chambers or sites in an array of reaction chambers or sites, and amplifying individual target polynucleotides within the reaction chambers or sites. Optionally, two or more target polynucleotides are partitioned into two or more reaction chambers or sites of the array, and the two or more partitioned target polynucleotides are amplified in parallel within their respective reaction chambers or sites. Optionally, at least two of the reaction chambers or sites each receive a single target polynucleotide during partitioning (one or more of the reaction chambers or sites may optionally receive zero or more target polynucleotides during partitioning). At least two target polynucleotides may be clonally amplified within their respective reaction chambers. The at least one reaction chamber comprising the target polynucleotide may be in fluid communication with at least one other reaction chamber comprising the target polynucleotide during amplification.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, devices, and kits) for nucleic acid amplification, comprising: (a) Partitioning at least two different polynucleotides into an array of reaction chambers by introducing a single polynucleotide into at least two reaction chambers in fluid communication with each other; and (b) forming at least two substantially monoclonal nucleic acid populations by amplifying the polynucleotides in the at least two reaction chambers. Typically, at least two reaction chambers are in fluid communication with each other during amplification.

In some embodiments, the present disclosure relates generally to methods (and related compositions, systems, devices, and kits) for nucleic acid amplification, comprising: (a) In an array of reaction chambers comprising first and second reaction chambers, partitioning a first template polynucleotide into the first reaction chamber and a second template polynucleotide into the second reaction chamber, and (b) forming at least two substantially monoclonal nucleic acid populations by clonally amplifying the first and second template polynucleotides within their respective reaction chambers, wherein a single polynucleotide is partitioned from a nucleic acid sample having a plurality of different polynucleotides. Optionally, the first and second reaction chambers comprise different portions of a single continuous liquid phase during amplification. For example, the first and second reaction chambers of the array may be in fluid communication during amplification.

In some embodiments, the present disclosure generally relates to methods for nucleic acid amplification, comprising (a) partitioning a different single polynucleotide into each of a plurality of reaction chambers, and (b) forming a population of monoclonal nucleic acids in each of the reaction chambers by amplifying the different single polynucleotides within the plurality of reaction chambers, wherein a single different polynucleotide is partitioned from a nucleic acid sample having a plurality of different polynucleotides.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification, comprising (a) partitioning at least two different polynucleotides into an array of reaction chambers by introducing a single polynucleotide into at least two reaction chambers in fluid communication with each other; and (b) forming at least two substantially monoclonal nucleic acid populations by amplifying the polynucleotides in the at least two reaction chambers.

In some embodiments, the method may further comprise introducing one or more supports (e.g., beads or particles, etc.) into at least one reaction chamber or site of the array. One or more supports may be introduced into at least one reaction chamber or site before, during or after the polynucleotides are dispensed into the array. In some embodiments, at least one reaction chamber or site of the array receives a single support. In some embodiments, a majority of the reaction chambers or sites receive a single support. In some embodiments, the support may be mixed with the polynucleotides and partitioned into the array with the polynucleotides prior to partitioning. At least one support may optionally be linked to a nucleic acid molecule comprising a primer sequence that is substantially complementary or substantially identical to a portion of a polynucleotide present in a reaction chamber or site during amplification. In some embodiments, at least one support comprises a nucleic acid molecule comprising a primer sequence that is substantially complementary or substantially identical to a portion of a target polynucleotide or template in a reaction chamber or well. In some embodiments, at least one support comprises a nucleic acid molecule comprising a primer sequence that is substantially complementary or substantially identical to at least a portion of another primer present in a reaction chamber or site during amplification.

In some embodiments, amplification may include introducing a reaction mixture into at least one reaction chamber or site of the array. Optionally, the reaction mixture is introduced into the reaction chamber or site before, during or after the partitioning of the polynucleotides into the array or the introduction of the support into the array. The reaction mixture, support and polynucleotides may be introduced or partitioned into the array in any order or in any combination. In some embodiments, at least one reaction chamber or site of the array receives a single support, a single polynucleotide, and a reaction mixture sufficient to support amplification of the polynucleotide within the reaction chamber or site.

In some embodiments, the method may comprise hybridizing at least a portion of the polynucleotide to the support by contacting the support with the polynucleotide under hybridization conditions. Hybridization may occur before, during, or after introduction of the support and/or polynucleotides into the reaction chamber or site of the array. In some embodiments, at least one support linked to a first primer sequence is introduced into at least one reaction chamber or site of the array, followed by introduction of a polynucleotide into the reaction chamber or site where the polynucleotide hybridizes to the support. Alternatively, the support may hybridize to an amplification product produced by amplification of a polynucleotide within a reaction chamber or site.

The reaction mixture may include any of the reaction mixtures and components described herein. In some embodiments, the reaction mixture may include any one or more of the following components: isothermal amplification reagents (e.g., one or more recombinases, helicases, i.e., related cofactors, polymerases, etc.), sizing agents, nucleotides, and the like.

In some embodiments, the present disclosure relates generally to methods (and related compositions, kits, systems, and devices) for nucleic acid amplification, comprising: (a) Introducing a first polynucleotide template and a first support into a first reaction chamber or site of an array of reaction chambers or sites, and introducing a second polynucleotide template and a second support into a second reaction chamber or site of the array, in any order or combination; and (b) clonally amplifying the first polynucleotide template on the first support within the first reaction chamber or site and clonally amplifying the second polynucleotide template on the second support within the second reaction chamber or site, with the first reaction chamber or site being in fluid communication with the second reaction chamber or site during amplification. Clonally amplifying may include generating a first support linked to a first amplicon from a first polynucleotide template and a second support linked to a second amplicon from a second polynucleotide template. Optionally, the first and second sites (or reaction chambers) comprise the same continuous liquid phase of the same reaction mixture during amplification. For example, the reaction mixture may comprise a single continuous liquid phase comprising the first and second polynucleotide templates and the first and second supports. The reaction mixture may be introduced into the reaction chambers or sites of the array before, during, or after the introduction of the polynucleotide template and/or support. In some embodiments, the disclosed methods further comprise introducing the reaction mixture into the first and second reaction chambers or sites after introducing the first and second polynucleotide templates and the first and second supports. In some embodiments, the reaction mixture includes a recombinase or helicase or a recombinase and a helicase. The recombinase may be derived from a myo-tail virus (e.g., uvsX), a bacterial, yeast or human recombinase or an analog thereof from another species. In some embodiments, the reaction mixture comprises a polymerase. In some embodiments, the reaction mixture comprises a sieving agent, such as polyacrylamide, agarose, or a cellulose polymer (e.g., HEC, CMC, or MC, or derivatives thereof). In some embodiments, the reaction mixture comprises a diffusion limiting agent.

In some embodiments, amplifying includes hybridizing a first primer binding sequence of a polynucleotide template to a first primer sequence of a first primer of a support to attach the polynucleotide template to the support or surface (e.g., particle or bead) having the first primer comprising the first primer sequence.

In embodiments wherein amplification is performed within a reaction chamber or site of the array, and embodiments wherein amplification is performed within a single reaction vessel, the surface or support optionally comprises at least a first primer comprising a first primer sequence. In some embodiments, one or more polynucleotide templates comprise a first primer binding sequence. The first primer binding sequence may be identical or substantially identical to the first primer sequence. Alternatively, the first primer binding sequence may be complementary or substantially complementary to the first primer sequence. In some embodiments, the first primer sequence and the first primer binding sequence do not exhibit substantial identity or complementarity, but are substantially identical or substantially complementary to another nucleotide sequence present in the reaction mixture. In such embodiments, amplification may include the formation of amplification reaction intermediates including nucleotide sequences that have significant identity or complementarity to the first primer sequence, the first primer binding sequence, or both.

In some embodiments, there are at least two different polynucleotide templates in the reaction mixture, and the amplification results in the formation of at least two different substantially monoclonal populations, each derived from the amplification of a single said polynucleotide template. In some embodiments, two or more of the at least two substantially monoclonal populations are attached to the same support or surface. Each of the two or more substantially monoclonal populations may be attached to a different unique location on the same support or surface. Alternatively, each of the two or more substantially monoclonal populations may be individually attached to a different support or surface. The separation of different monoclonal populations to different supports or surfaces may be advantageous in applications where it is desirable to separate populations prior to analysis. In some embodiments, the support or surface is part of a bead or particle, which may be spherical or spheroid in shape. In some embodiments, the support or surface forms part of a two-dimensional or three-dimensional array.

In some embodiments, the disclosed methods for nucleic acid amplification can be performed when a sieving agent or a diffusion reducing agent is included in the reaction mixture. These agents can increase the total number and/or proportion (e.g., percentage) of monoclonal populations formed during amplification. In some embodiments, the method comprises using a reaction mixture that provides increased yields of monoclonal or substantially monoclonal amplicons relative to conventional reaction mixtures.

In some embodiments, the method comprises amplifying by partially denaturing the template. For example, amplification includes template walking. For example, the template to be amplified may include a linker sequence comprising a primer binding site that has a relatively low Tm compared to the template as a whole. In some embodiments, the amplification is performed at a temperature substantially above the Tm of the linker sequence and substantially below the Tm of the template, as described in more detail herein. In some embodiments, the amplification is performed at a temperature at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ below the Tm of the nucleic acid template. In some embodiments, the amplification is performed at a temperature above (e.g., at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, or 50 ℃ or higher) the Tm of the first primer, the second primer, or the first and second primers.

In some embodiments, the reaction mixture may optionally include any one or more of the following components: (a) Optionally one or more supports comprising at least a first primer sequence; (2) a recombinase; (3) a polymerase; (4) a diffusion limiting agent; (5) a sieving agent; (6) crowding agents; (7) an ATP regeneration system; (8) single chain binding protein (SSBP); and (8) a recombinase cofactor, such as a recombinase-loaded protein. In some embodiments, the yield of a monoclonal or substantially monoclonal population is increased by amplifying (e.g., by ligating one amplification primer to the surface) the polynucleotide template on the surface in the presence of a diffusion limiting agent and/or a sieving agent. Diffusion limiters or sieving agents can provide increased yields of monoclonal populations by reducing diffusion or migration of amplified product polynucleotides off the surface during amplification.

In some embodiments, the reaction mixture includes one or more isothermal amplification reagents. Such reagents may include, for example, a recombinase or helicase.

In some embodiments, the method for nucleic acid amplification may be performed using an enzyme that catalyzes homologous recombination, e.g., an enzyme that may bind to a first primer to form a complex or may catalyze strand invasion or may form a D-loop structure. In some embodiments, the enzyme that catalyzes homologous recombination comprises a recombinase.

In some embodiments, the amplification conditions comprise isothermal conditions or thermocycling conditions.

In some embodiments, a method for nucleic acid amplification comprises: (a) Forming a reaction mixture comprising a single continuous liquid phase comprising (i) an enzyme that catalyzes homologous recombination, (ii) one or more surfaces, and (iii) a plurality of different polynucleotides; and (b) subjecting the reaction mixture to conditions suitable for nucleic acid amplification.

In some embodiments, a method for nucleic acid amplification comprises: (a) Forming a reaction mixture comprising a single continuous liquid phase comprising (i) an enzyme that catalyzes homologous recombination, (ii) one or more beads each linked to a plurality of first primers, and (iii) a plurality of different polynucleotides; (b) Two or more substantially monoclonal amplified nucleic acid populations are formed by subjecting the reaction mixture to amplification conditions. Amplification conditions may include isothermal or thermocycling conditions. In some embodiments, the first primer can hybridize to at least a portion of the polynucleotide.

In some embodiments, the present disclosure relates generally to methods for nucleic acid amplification, comprising (a) forming a reaction mixture comprising a single continuous liquid phase comprising one or more supports (or surfaces), a plurality of polynucleotides, and a recombinase enzyme; (b) At least two of the plurality of different polynucleotides are clonally amplified on at least one support (or surface) by subjecting the reaction mixture to amplification conditions. In some embodiments, the amplification conditions may include isothermal or thermocycling amplification conditions. The reaction mixture may optionally include a recombinase. In some embodiments, the reaction mixture includes a polymerase. In some embodiments, the reaction mixture includes primers, which may be in solution. Optionally, at least one of the support or surface may comprise a primer.

In some embodiments, the present disclosure generally relates to methods for nucleic acid amplification, comprising (a) forming a reaction mixture comprising a single continuous liquid phase comprising (i) a recombinase, (ii) a plurality of beads linked to one or more first primers comprising a first primer sequence, and (iii) a plurality of different polynucleotide templates; (b) Hybridizing at least one of the first primers to at least one of a plurality of different polynucleotide templates; (c) Subjecting the reaction mixture to nucleic acid amplification conditions and generating at least one substantially monoclonal polynucleotide population by amplifying at least one polynucleotide template to form at least a first amplification population. In some embodiments, at least 30%, 90% of the polynucleotides in at least one substantially monoclonal population are substantially identical (or substantially complementary) to at least one polynucleotide template originally present in the reaction mixture. In some embodiments, at least a portion of the first amplification population is attached to one bead of the plurality of beads.

In some embodiments, forming the reaction mixture in step (a) comprises: a nucleoprotein complex is formed by contacting a recombinase with at least one of a plurality of first primers linked to a plurality of beads.

In some embodiments, subjecting the reaction mixture to nucleic acid amplification conditions in step (b) comprises performing a nucleotide polymerization reaction. For example, nucleotide polymerization may include incorporating nucleotides into the first primer sequence, optionally when the first primer sequence hybridizes to a polynucleotide template in the reaction mixture.

In some embodiments, subjecting the reaction mixture to nucleic acid amplification conditions comprises contacting the first primer with a polynucleotide template, a recombinase, a polymerase, and nucleotides in any order or in any combination.

In some embodiments, the nucleic acid amplification conditions comprise repeating such a cycle: forming a nucleoprotein complex comprising a recombinase, at least a portion of the first primer, and at least a portion of the first polynucleotide template, and contacting the nucleoprotein complex with a polymerase that catalyzes the incorporation of one or more nucleotides to the first primer.

In some embodiments, a cycling nucleic acid amplification reaction can be performed to produce a plurality of beads each linked to a substantially monoclonal polynucleotide population.

In some embodiments, the method for nucleic acid amplification may be performed under isothermal conditions or thermocycling conditions.

In some embodiments, the plurality of different polynucleotides may be single-stranded or double-stranded polynucleotides. In some embodiments, thermal or chemical denaturation of the double-stranded polynucleotide is not necessary, as the recombinase can produce localized strand denaturation by catalyzing strand invasion.

In some embodiments, the method for nucleic acid amplification may be performed in a single reaction vessel. In some embodiments, the nucleic acid amplification reaction may be performed in a single reaction vessel comprising a single continuous liquid phase. For example, a single continuous liquid phase may comprise an amplification mixture comprising a plurality of beads each having a plurality of first primers attached thereto, a plurality of different polynucleotides, and a plurality of recombinases. In some embodiments, the amplification mixture may further comprise a polymerase and a plurality of nucleotides. In some embodiments, the amplification mixture may further comprise ATP, nucleotides, and cofactors. Non-limiting examples of a single reaction vessel include a tube, a well, or similar structure.

In some embodiments, the polynucleotide and reagent may be placed into the reaction vessel in any order, including sequentially or substantially simultaneously or a combination of both. In some embodiments, the reagent comprises a bead, recombinase, polymerase, nucleotide, ATP, divalent cation, and cofactor linked to a plurality of first primers.

In some embodiments, the method for nucleic acid amplification may be performed in a single continuous liquid phase. A single continuous liquid phase may include any liquid phase in which any given portion or region of the single continuous liquid phase is in fluid communication with any other portion or region of the same single continuous liquid phase. Typically, components dissolved or suspended in a single continuous liquid phase can freely diffuse or migrate to any other point in the liquid phase. In some embodiments, however, the single continuous liquid phase may include a diffusion limiting agent that slows the diffusion rate in the single continuous liquid phase. An exemplary embodiment of a single continuous liquid phase is a single aqueous droplet in a water-in-oil emulsion; in such emulsions, each droplet will form a separate phase; the two droplets may combine to form a single phase.

In some embodiments, the single continuous liquid phase consists essentially of a single aqueous phase. In some embodiments, the single continuous liquid phase lacks a non-aqueous phase; for example, the continuous liquid phase does not include oil or an organic solvent. In some embodiments, the plurality of nucleic acid amplification reactions occur in the aqueous phase of a single reaction vessel. In some embodiments, a single continuous liquid phase does not partition where multiple nucleic acid amplification reactions occur in a single reaction vessel.

In some embodiments, the method for nucleic acid amplification may be performed in a water-in-oil emulsion that provides partitioning.

When nucleic acid amplification is performed using multiple polynucleotide templates, clonal amplification using conventional amplification methods typically relies on techniques such as partitioning the reaction mixture into separate fractions or components that are not in fluid communication with each other to maintain clonality and prevent cross-contamination of different amplification populations and to maintain adequate yields of monoclonal amplification products. Using such conventional amplification methods, clonally amplifying the polynucleotide templates within the same reaction mixture without partitioning or partitioning of the reaction mixture into separate compartments or vessels is generally not feasible, as any polynucleotides (including templates and/or amplification products) within the reaction mixture will tend to migrate randomly through the mixture due to diffusion and/or brownian motion during such amplification. Such diffusion or migration generally increases the occurrence of polyclonal amplifications, which will result in very few, if any, monoclonal populations.

One suitable technique used in conventional amplification methods to reduce the generation of polyclonal populations uses a physical barrier to separate individual amplification reactions into discrete compartments. For example, emulsion PCR uses a water-in-oil microreaction vessel in which the oil phase comprises a number of separate, i.e., discrete, aqueous reaction compartments. Each compartment serves as a separate amplification reaction vessel so that the entire emulsion is able to support a number of separate amplification reactions in separate (discrete) liquid phases of a single reaction vessel (e.g., eppendorf tube or well). Similarly, an amplification "master mix" can be prepared and dispensed into separate reaction chambers (e.g., arrays of wells) resulting in a set of discrete and separate phases, each defining a separate amplification reaction. Such separate phases may be further blocked from each other prior to amplification. Such a closure may be used to prevent cross-contamination between parallel and separate reactions. An exemplary form of closure may include the use of a lid or phase barrier (e.g., a mineral oil layer over an aqueous reaction) to divide the PCR reaction into individual and discrete compartments between which transfer of reaction components does not occur.

Other techniques to prevent cross-contamination and reduce polyclonality rely on the immobilization of one or more reaction components (e.g., one or more templates and/or primers) during amplification to prevent cross-contamination of the amplified reaction products and their resulting reduction in monoclonality. One such example includes bridge PCR, in which all primers (e.g., forward and reverse primers) required for amplification are attached to the surface of a matrix support. In addition to such immobilization, additional immobilization components may be included in the reaction mixture. For example, the polynucleotide templates and/or amplification primers may be suspended in a gel or other matrix during amplification to prevent migration of amplification reaction products from the synthesis site. Such gels and matrices typically need to be removed later, which requires the use of an appropriate "melting" or other recovery step, thereby losing yield.

In some embodiments, the present disclosure provides methods for conducting substantially monoclonal amplification of multiple polynucleotide templates in parallel in a single continuous liquid phase of a reaction mixture without the need for partitioning or immobilization of multiple reaction components (e.g., two primers) during amplification. Alternatively, the mixture of polynucleotide templates in solution may be directly contacted with the amplification reaction components and the appropriate surface or support with the first primer attached thereto, and other components required for amplification may be provided in the same continuous liquid phase, including the polymerase, one or more types of nucleotides, and optionally the second primer. In some embodiments, the reaction mixture further comprises a recombinase. Optionally, the reaction mixture further comprises at least one agent selected from the group consisting of diffusion limiting agents, sieving agents, and crowding agents. Examples of amplification mixtures suitable for effecting monoclonal amplification of templates contained in a single continuous liquid phase are further described herein. Optionally, different templates may be amplified on different locations on a single surface or support, or different templates may be amplified on different surfaces or different supports within the same reaction mixture.

In some embodiments, the reaction mixture may include one or more sieving agents. Screening agents optionally include any compound that can provide a physical barrier to migration of a polynucleotide template or its corresponding amplification product. (migration may include any movement of template or amplification product within the reaction mixture; diffusion includes forms of migration involving movement along a concentration gradient). In some embodiments, the sieving agent comprises any compound that can provide a matrix with a plurality of pores that are small enough to reduce movement of the nucleic acid synthesis reaction mixture or any one or more specific components of the nucleic acid reaction mixture.

In some embodiments, the sieving agent provides a molecular sieve. For example, a sieving agent can reduce movement of polynucleotides (or polynucleotides associated with a surface or bead) through a reaction mixture comprising the sieving agent. The sieving agent may optionally have small pores.

The inclusion of a sieving agent may be advantageous when two or more template polynucleotides are clonally amplified within a single continuous liquid phase of the reaction mixture. For example, the sieving agent may prevent or slow the diffusion of the template or amplified polynucleotides produced by replication of at least some portion of the template within the reaction mixture, thereby preventing the formation of polyclonal contaminants without the need to partition the reaction mixture by physical methods or encapsulation methods (e.g., emulsion) during amplification. Such a method of clonally amplifying templates within a single continuous liquid phase of a single reaction mixture without partitioning greatly reduces the cost, time and effort associated with generating libraries suitable for use in high throughput methods such as digital PCR, next generation sequencing, and the like.

In some embodiments, the average pore size of the sieving agent is such that movement of the target component (e.g., polynucleotide) within the reaction mixture is selectively impeded or prevented. In one example, the sieving agent comprises any compound that can provide a matrix having a plurality of pores that are small enough to slow or impede the movement of polynucleotides through a reaction mixture comprising the sieving agent. Thus, the sieving agent may reduce brownian motion of the polynucleotide.

In some embodiments, the sieving agent selectively acts to block migration of molecules having an average molecular size or weight above a particular threshold or range, while not blocking migration of other molecules having an average molecular size or weight below the threshold or range.

In some embodiments, the sieving agent selectively acts to block migration of molecules having an average molecular size or weight below a particular threshold or range, while not blocking migration of other molecules having an average molecular size or weight above the threshold or range.

In some embodiments, the sieving agent may be selected to selectively hinder, slow, reduce, or prevent movement of the polynucleotide through the reaction mixture, but is large enough to allow movement of smaller components (e.g., cations, nucleotides, ATP, and cofactors) through the reaction mixture. In some embodiments, the sieving agent has an average pore size or range of average pore sizes that can be adjusted by increasing or decreasing the concentration of the sieving agent. For example, the molecular weight, intrinsic viscosity, and concentration of the sieving agent (or combination of sieving agents) may be selected to prepare a nucleic acid reaction mixture in a particular solvent (e.g., water) to produce such a matrix: it has a desired ability to prevent migration of a target polynucleotide of a particular size or length, or a desired average pore size or viscosity. In some embodiments, the sieving agent can reduce the overall flow rate by increasing the viscosity of the nucleic acid reaction mixture. In some embodiments, the sieving agent may be water soluble. In some embodiments, a matrix having a plurality of pores may be prepared by mixing a sieving agent with a solvent (e.g., an aqueous solvent, such as water). In some embodiments, the sieving agent does not interfere with the formation of the recombinase nucleoprotein complex or with the polymerization of nucleotides.

In some embodiments, the present disclosure generally relates to methods for performing a nucleic acid amplification reaction, comprising generating two or more substantially monoclonal populations by amplifying a target polynucleotide on a surface or support in the presence of one or more sieving agents, optionally in the presence of a recombinase, a polymerase, or any other suitable agent capable of catalyzing or promoting nucleic acid amplification.

In some embodiments, inclusion of a sizing agent in the reaction mixture may reduce movement of the polynucleotide away from a given support or surface (e.g., reduce shedding) and may increase the likelihood that the polynucleotide will hybridize to the support or surface and provide an initiation site for nucleotide polymerization, thereby increasing the proportion of substantially monoclonal amplicon generated during the amplification reaction.

In some embodiments, amplifying comprises amplifying a plurality of different polynucleotide templates on a plurality of different bead supports in the presence of a sieving agent, and recovering a percentage of substantially monoclonal bead supports, each such substantially monoclonal bead support comprising a bead support linked to a substantially monoclonal polynucleotide population. In some embodiments, the percentage of substantially monoclonal bead support recovered is substantially greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 90% or 95% of the total amplified bead support recovered from the reaction mixture (i.e., the total bead support comprising the polyclonal or monoclonal population). In some embodiments, the percentage of substantially monoclonal bead support recovered is substantially higher than the percentage of substantially monoclonal bead support recovered after amplification in the absence of a sieving agent but under otherwise substantially similar or identical amplification conditions.

In some embodiments, the sieving agent comprises a polymeric compound. In some embodiments, the sieving agent comprises a crosslinked or non-crosslinked polymeric compound. By way of non-limiting example, the sieving agent may include polysaccharides, polypeptides, organic polymers, and the like.

In some embodiments, the sieving agent comprises a linear or branched polymer. In some embodiments, the sieving agent comprises a dotted or neutral polymer.

In some embodiments, the sieving agent may comprise a mixture of one or more polymers each having an average molecular weight and viscosity.

In some embodiments, the sieving agent comprises a polymer having an average molecular weight of about 10,000-2,000,000 or about 12,000 to 95,000 or about 13,000 to 95,000.

In some embodiments, the sieving agent may exhibit an average viscosity ranging from about 5 centipoise to about 15,000 centipoise measured at about 25 ℃ when dissolved in water at 2 weight percent, or from about 10 centipoise to about 10,000 centipoise measured at about 25 ℃ as a 2% aqueous solution, or from about 15 centipoise to about 5,000 centipoise measured at about 25 ℃ as a 2% aqueous solution.

In some embodiments, the sieving agent comprises about 25 to about 1,5000km _v Or about 75 to 1,000kM _v Or about 85 to 800kM _v Viscosity average molecular weight (M) _v ). In some embodiments, the reaction mixture includes about 0.1 to about 20% weight/volume or about 1-10% w/v or about 2-5% w/v of the sieving agent.

In some embodiments, the sieving agent comprises an acrylamide polymer such as polyacrylamide.

In some embodiments, the sieving agent comprises a polymer of one or more amino acids. For example, the sieving agent may include polylysine, polyglutamic acid, actin, myosin, keratin, tropomyosin (tropmyosin), and the like. In some embodiments, the sieving agent may comprise a derivative of any of these polypeptides.

In some embodiments, the sieving agent comprises a polysaccharide polymer. In some embodiments, the sieving agent comprises a polymer of glucose or galactose. In some embodiments, the sieving agent comprises one or more polymers selected from cellulose, dextran, starch, glycogen, agar, chitin, pectin, or agarose. In some embodiments, the sieving agent comprises a glucopyranose polymer.

In some embodiments, the sieving agent comprises a polymer having one or more groups that are polar or charged under the amplification reaction conditions. For example, the polymer may comprise one or more cationic groups, one or more anionic groups, or both. In some embodiments, the sieving agent is a polysaccharide comprising one or more charged groups. In some embodiments, the sieving agent is a polysaccharide comprising one or more carboxyl groups that are or tend to be negatively charged under amplification reaction conditions. For example, the sieving agent may comprise a carboxymethyl cellulose (CMC) polymer. In some embodiments, the sieving agent may comprise spermine and/or spermidine. In some embodiments, the sieving agent comprises polylysine and/or polyarginine. For example, the sieving agent may include poly-L-lysine, poly-D-glutamic acid, and the like. In some embodiments, the sieving agent comprises one or more histones or histone-nucleic acid complexes or derivatives thereof. Histones are highly basic proteins that are capable of binding nucleic acids and include proteins H1, H2A, H2B, H3 and H4. In some embodiments, the histone is modified, e.g., by methylation, acetylation, phosphorylation, ubiquitination, SUMO methylation, citrullination, ribosylation (including ADP-ribosylation), and the like.

In some embodiments, the sieving agent comprises a polymer, including a chemically substituted polymer. The polymer may include reactive groups (e.g., the reactive groups may react with suitable substituents to produce a substituted polymer). In some embodiments, the polymer comprises a fluorescent-, carboxyl-, amino-, or alkoxy-substituted polymer. In some embodiments, the polymer is modified by methylation, acetylation, phosphorylation, ubiquitination, carboxylation, and the like. The substituents may include charged groups, such as anionic or cationic groups, substitution of which into the polymer chain may result in the production of charged polymers. The degree of substitution may vary from about 0.2 to about 1.0 derivative/monomer units, typically from about 0.4 to about 1.0, more typically from about 0.6 to about 0.95.

In some embodiments, the sieving agent comprises a cellulose derivative, such as sodium carboxymethyl cellulose, sodium carboxymethyl 2-hydroxyethyl cellulose, methyl cellulose, hydroxyethyl cellulose, 2-hydroxypropyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose, (hydroxypropyl) methyl cellulose, or hydroxyethyl ethyl cellulose, or a mixture comprising any one or more of such polymers.

In some embodiments, the nucleic acid reaction mixture includes a mixture of different sieving agents, such as a mixture of different cellulose derivatives, starches, polyacrylamides, and the like.

In some embodiments, the sieving agent comprises one or more composite polymers comprising sub-portions of any two different polymers (including any of the polymers described herein). For example, the complexing polymer may comprise a polysaccharide polymer linked to a polynucleotide polymer, such as a polysaccharide linked to DNA or RNA. In some embodiments, the sieving agent may comprise a polymer comprising a cellulose moiety and a nucleic acid moiety, such as DNA-cellulose. In other embodiments, the complexing polymer may comprise polyacrylamide linked to the polynucleotide and/or polypeptide. The inclusion of such complexing polymers in the reaction mixture may be used to further hinder the movement of the target polynucleotide through the reaction mixture.

In some embodiments, the sieving agent may comprise a polymer that has been first contacted or reacted with a suitable crosslinking agent. For example, the sieving agent may comprise acrylamide that has been reacted with bisacrylamide and/or bis (acryloyl) cystamine.

In some embodiments, the reaction mixture includes at least one diffusion reducing agent. In some embodiments, the diffusion reducing agent comprises any compound that reduces the migration of a polynucleotide from a region of higher concentration to a region having a lower concentration. In some embodiments, the diffusion reducing agent comprises any compound that reduces migration of any component (regardless of size) of the nucleic acid amplification reaction. In some embodiments, the components of the nucleic acid amplification reaction include beads/primers, polynucleotides, recombinases, polymerases, nucleotides, ATP, and/or cofactors.

It should be noted that the concepts of sieving agent and diffusion reducing agent are not necessarily mutually exclusive; sieving agents often effectively reduce diffusion of target compounds through the reaction mixture, while diffusion reducing agents often can have a sieving effect on the reaction components. In some embodiments, the same compound or reaction mixture additive may be used as a sieving agent and/or a diffusion reducing agent. Any of the screening agents disclosed herein may be capable of hooking to act as a diffusion reducing agent in some embodiments and vice versa.

In some embodiments, the diffusion reducing agent and/or sieving agent comprises polyacrylamide, agar, agarose, or a cellulosic polymer such as Hydroxyethylcellulose (HEC), methylcellulose (MC), or carboxymethylcellulose (CMC).

In some embodiments, the sieving agent and/or diffusion reducing agent is included in the reaction mixture at a concentration of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 74%, 90% or 95% w/v (weight of agent/unit volume of reaction mixture).

In some embodiments, the reaction mixture includes at least one crowding reagent. For example, crowding reagents can increase the concentration of one or more components in a nucleic acid amplification reaction by creating a crowded reaction environment. In some embodiments, the reaction mixture includes a sieving agent and a crowding agent.

In some embodiments, a method for nucleic acid amplification includes one or more surfaces. In some embodiments, the surface may have a plurality of first primers attached thereto, the first primers of the plurality having a common first primer sequence.

In some embodiments, the surface may be an outer or uppermost layer or boundary of the object. In some embodiments, the surface may be inside the boundary of the object.

In some embodiments, the reaction mixture includes a plurality of different surfaces, e.g., the reaction mixture may include a plurality of beads (e.g., particles, nanoparticles, microparticles, etc.) and at least two different polynucleotide templates may be clonally amplified on the different surfaces, thereby forming at least two different surfaces, each of which is attached to an amplicon. In some embodiments, the reaction mixture comprises a single surface (e.g., a surface of a slide or an array of reaction chambers) and at least two different polynucleotide templates are amplified at two different regions or locations on the surface, thereby forming a single surface that is linked to two or more amplicons.

In some embodiments, the surface may be porous, semi-porous, or non-porous. In some embodiments, the surface may be a planar surface as well as a concave surface, a convex surface, or any combination thereof. In some embodiments, the surface may be a bead, particle, microparticle, sphere, filter, flow cell, well, trench, groove receptacle, gel, or inner wall of a capillary. In some embodiments, the surface comprises a capillary, a groove, a hole, a groove, an inner wall of a container. In some embodiments, the surface may include texture (e.g., etched, cavitated, small holes, three-dimensional scaffolds, or ridges).

In some embodiments, the particles may have a sphere, hemisphere, cylinder, barrel, ring, rod, disk, cone, triangle, cube, polygon. Tubular, linear or irregular.

In some embodiments, the surface may be prepared from any material including glass, borosilicate glass, silica, quartz, fused silica, mica, polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramic, silicon, semiconductors, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of gold, silver, aluminum, or diamond).

In some embodiments, the film may be magnetic or paramagnetic beads (e.g., magnetic or paramagnetic nanoparticles or microparticles). In some embodiments, the paramagnetic microparticles may be paramagnetic beads (e.g., dynabeads from Invitrogen, carlsbad, calif.) with streptavidin attached ^TM M-270). The particles may have an iron core or comprise a hydrogel or agarose (e.g. Sepharose ^TM )。

In some embodiments, a plurality of first primers may be attached to the surface. The surface may be coated with an acrylamide, carboxyl or amine compound to bind nucleic acid (e.g., the first primer). In some embodiments, amino modified nucleic acids (e.g., primers) can be attached to a carboxylic acid coated surface. In some embodiments, the amino modified nucleic acid may be reacted with EDC (or EDAC) to attach to a carboxylic acid coated surface (with or without NHS). The first primer may be immobilized to an acrylamide compound coating on the surface. The particles may be coated with an avidin-like compound (e.g., streptavidin) to bind the biotinylated nucleic acid.

In some embodiments, the surface comprises the surface of a bead. In some embodiments, the beads comprise a polymeric material. For example, the beads include gels, hydrogels, or acrylamide polymers. The beads may be porous. The particles may have cavities or pores, or may comprise a three-dimensional scaffold. In some embodiments, the particles may be Ion Sphere ^TM And (3) particles.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) comprise immobilizing one or more nucleic acid templates on one or more supports. The nucleic acid may be immobilized on the solid support by any method including, but not limited to, physical adsorption, by ionic or covalent bond formation, or a combination thereof. The solid support may comprise polymeric, glass or metallic materials. Examples of solid supports include membranes, planar surfaces, microwell plates, beads, filters, test strips, slides, coverslips, and test tubes. Meaning any solid phase material on which the oligomer is synthesized, attached, linked or otherwise immobilized. The support may optionally comprise a "resin", "phase", "surface" and "support". The support may be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyvinylfluoride, polyethyleneoxy (polyethyleneoxy) and polyacrylamide and copolymers and grafts thereof. The support may also be inorganic, such as glass, silica, controlled Pore Glass (CPG) or inverted silica. The support structure may be in the form of beads, spheres, particles, granules, gels or surfaces. The surface may be planar, substantially planar or non-planar. The support may be porous or non-porous and may have swelling or non-swelling properties. The support may be shaped to include one or more holes, depressions or other reservoirs, containers, features or locations. Multiple supports may be arranged at different locations in the array. The support is optionally addressable (e.g., for robotic delivery of reagents), or by detection means including scanning by laser irradiation and confocal or deflected light collection. The amplification support (e.g., a bead) may be placed in or on another support (e.g., within a well of a second support).

In embodiments, the solid support is a "microparticle," "bead," "microbead," or the like (optionally but not necessarily spherical in shape) having a minimum cross-sectional length (e.g., diameter) of 50 microns or less, preferably 10 microns or less, 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3, or 0.4 microns, or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers). Microparticles (e.g., dynabead from Dynal, oslo, norway) may be made from a variety of inorganic or organic materials including, but not limited to, glass (e.g., controlled pore glass), silica, zirconium, crosslinked polystyrene, polyacrylate, polymethyl methacrylate, titanium dioxide, latex, polystyrene, and the like. Magnetization can aid in the collection and concentration of the microparticle-linked reagents (e.g., polynucleotides or ligases) after amplification, and can also aid in additional steps (e.g., washing, reagent removal, etc.). In certain embodiments of the invention, populations of particles having different shape sizes and/or colors may be used. The microparticles may optionally be encoded, for example, with quantum dots so that such microparticles may be individually or uniquely identified.

In some embodiments, the bead surface may be functionalized to ligate a plurality of first primers. In some embodiments, the beads can be any size that can fit into a reaction chamber. For example, a bead can be assembled into a reaction chamber. In some embodiments, more than one bead may be assembled into a reaction chamber. In some embodiments, the minimum cross-sectional length (e.g., diameter) of the beads can be about 50 microns or less, or about 10 microns or less, or about 3 microns or less, about 1 micron or less, about 0.5 microns or less, such as about 0.1, 0.2, 0.3, or 0.4 microns, or less (e.g., less than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers).

In some embodiments, the bead may have a plurality of one or more different primer sequences attached. In some embodiments, the bead may have a plurality of one primer sequence attached, or may have a plurality of two or more different primer sequences attached. In some embodiments, the bead may have at least 1,000 primers attached, or about 1,000-10,000 primers, or about 10,000-50,000 primers, or about 50,000-75,000 primers, or about 75,000-100,000 primers or more.

In some embodiments, the reaction mixture includes a recombinase. The recombinase may include any agent capable of inducing a recombination event or increasing the frequency of occurrence of a recombination event. Recombination events include any event in which two different polynucleotide strands recombine with each other. Recombination may include homologous recombination. The recombinase may be an enzyme or a genetically engineered derivative thereof. The recombinase may optionally be associated (e.g., bound) with a single stranded oligonucleotide (e.g., a first primer). In some embodiments, the enzyme that catalyzes homologous recombination may form a nucleoprotein complex by binding single-stranded oligonucleotides. In some embodiments, the homologous recombinase enzyme as part of the nucleoprotein complex can bind to the homologous portion of the double-stranded polynucleotide. In some embodiments, the homologous portion of the polynucleotide can hybridize to at least a portion of the first primer. In a certain embodiment, the homologous portion of the polynucleotide may be partially or fully complementary to at least a portion of the first primer.

In some embodiments, homologous recombinases can catalyze strand invasion by forming a nucleoprotein complex and binding to homologous portions of the double-stranded polynucleotide to form a recombinant intermediate with a triple-stranded structure (D-loop formation) (U.S. Pat. No. 5,223,414 to Zarling, U.S. Pat. nos. 5,273,881 and 5,670,316 to sena, and U.S. Pat. nos. 7,270,981, 7,399,590, 7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000, 8,062,850, and 8071308). In some embodiments, the homologous recombinase comprises a wild-type, mutant, recombinant, fusion, or fragment thereof. In some embodiments, homologous recombinases include enzymes from any organism including the myoviridae (e.g., uvsX from bacteriophage T4, RB69, etc.), escherichia coli (e.g., recA), or humans (e.g., RAD 51).

In some embodiments, the method for nucleic acid amplification comprises one or more accessory proteins. For example, accessory proteins can improve the activity of a recombinase (U.S. Pat. No. 8,071,308 to Piepenburg, etc.). In some embodiments, the accessory protein may bind to a single strand of a nucleic acid or a recombinase may be loaded on the nucleic acid. In some embodiments, the helper protein comprises a wild type, mutant, recombinant, fusion, or fragment thereof. In some embodiments, the accessory protein may be derived from any combination of the same or different species as the recombinase used to perform the nucleic acid amplification reaction. The helper protein may be derived from any bacteriophage including a myotail virus bacteriophage. Examples of myotail virus phages include T4, T2, T6, rb69, aeh, KVP40, acinetobacter phage 133, aeromonas phage 65, blue-green algae phage P-SSM2, blue-green algae phage PSSM4, blue-green algae phage S-PM2, rb14, rb32, aeromonas phage 25, vibrio phage nt-1, phi-1, rb16, rb43, phage 31, phage 44RR2.8T, rb49, phage Rb3, and phage LZ2. The accessory protein may be derived from any bacterial species including E.coli, sulfolobus (e.g., sulfolobus) or Methanococcus (e.g., methanococcus jannaschii).

In some embodiments, the method for nucleic acid amplification may comprise a single-stranded binding protein. Single-chain binding proteins include myotail virus gp32 (e.g., T4 or RB 69), sso SSB from sulfolobus solfataricus, mjA SSB from Methanococcus jannaschii, or E.coli SSB protein.

In some embodiments, methods for nucleic acid amplification may include proteins that may increase recombinase loading on a nucleic acid. For example, recombinase-loaded proteins include the UsvY protein (U.S. patent 8,071,308 to piebenburg).

In some embodiments, the method for nucleic acid amplification may include at least one nucleic acid-binding protein, including a protein that untwists double-stranded nucleic acids (e.g., helicase).

In some embodiments, the method for nucleic acid amplification may include at least one cofactor for recombinase or polymerase activity. In some embodiments, the cofactor comprises one or more divalent cations. Examples of divalent cations include magnesium, manganese, and calcium.

In some embodiments, the nucleic acid amplification reaction may be pre-incubated under conditions that inhibit premature initiation of the reaction. For example, one or more components of the nucleic acid amplification reaction may be captured from the reaction vessel to prevent premature initiation of the reaction. To initiate the reaction, divalent cations (e.g., magnesium or manganese) may be added. In another example, the nucleic acid amplification reaction may be pre-incubated at a temperature that inhibits enzymatic activity. The reaction may be pre-incubated at about 0-15℃or about 15-25℃to inhibit premature initiation of the reaction. The reaction may then be incubated at a higher temperature to initiate the enzymatic activity.

In some embodiments, the method for nucleic acid amplification may include at least one cofactor for recombinase assembly on a nucleic acid or for homologous nucleic acid pairing. In some embodiments, the cofactor comprises any form of ATP including ATP and ATP γs.

In some embodiments, the method for nucleic acid amplification may include at least one cofactor that produces ATP. For example, cofactors include an enzyme system that converts ADP to ATP. In some embodiments, the cofactors include phosphocreatine and creatine kinase.

In some embodiments, any of the nucleic acid amplification methods disclosed herein can be performed under isothermal or substantially isothermal amplification conditions or can include steps performed under such conditions. In some embodiments, isothermal amplification conditions include nucleic acid amplification reactions that undergo such temperature changes: the temperature change is limited to a limited range during at least a portion of the amplification (or the entire amplification process), including, for example, a temperature change of equal to or less than about 20 ℃, or about 10 ℃, or about 5 ℃, or about 1-5 ℃, or about 0.1-1 ℃, or less than about 0.1 ℃.

In some embodiments, the isothermal nucleic acid amplification reaction may be performed for about 2, 5, 10, 15, 20, 30, 40, 50, 60, or 120 minutes.

In some embodiments, the isothermal nucleic acid amplification reaction may be performed at about 15-25 ℃, or about 25-35 ℃, or about 35-40 ℃, or about 40-45 ℃, or about 45-50 ℃, or about 50-55 ℃, or about 55-60 ℃.

In some embodiments, the method for nucleic acid amplification comprises a plurality of different polynucleotides. In some embodiments, the plurality of different polynucleotides comprises single-stranded or double-stranded polynucleotidesAn acid or a mixture of both. In some embodiments, the plurality of different polynucleotides includes polynucleotides having the same or different sequences. In some embodiments, the plurality of different polynucleotides comprises polynucleotides having the same or different lengths. In some embodiments, the plurality of different polynucleotides comprises about 2 to 10, or about 10 to 50, or about 50 to 100, or about 100 to 500, or about 500 to 1,000, or about 1,000 to 5,000, or about 10 ³ –10 ⁶ Or about 10 ⁶ -10 ¹⁰ One or more different polynucleotides. In some embodiments, the plurality of different polynucleotides comprises polymers of deoxynucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, the plurality of different polynucleotides includes naturally occurring, synthetic, recombinant, cloned, amplified, unamplified, or archived (e.g., preserved) forms. In some embodiments, the plurality of different polynucleotides comprises DNA, cDNA, RNA or chimeric RNA/DNA and nucleic acid analogs.

In some embodiments, the plurality of different polynucleotide templates may include double-stranded polynucleotide library constructs having one or both ends linked to a nucleic acid linker sequence. For example, a polynucleotide library construct may comprise a first and a second end, wherein the first end is linked to a first nucleic acid linker. The polynucleotide library construct may further comprise a second end linked to a second nucleic acid linker. The first and second linkers may have the same or different sequences. In some embodiments, at least a portion of the first or second nucleic acid adaptors (i.e., as part of a polynucleotide library construct) can be hybridized to the first primer. In some embodiments, the homologous recombinase as part of a nucleoprotein complex can be associated with a polynucleotide library construct having a first or second nucleic acid linker sequence.

In some embodiments, the polynucleotide library constructs may be suitable for use in any type of sequencing platform including chemical degradation, chain termination, sequencing by synthesis, pyrophosphate, massively parallel, ion sensitive, and single molecule platforms.

In some embodiments, the method for nucleic acid amplification comprises diluting the amount of polynucleotide reacted with a bead (e.g., a bead having a plurality of first primers attached) to reduce the percentage of beads reacted with more than one polynucleotide. In some embodiments, the nucleic acid amplification reaction can be performed using a polynucleotide to bead ratio selected to optimize the percentage of beads having a population of monoclonal polynucleotides attached thereto. For example, the nucleic acid amplification reaction may be performed at any polynucleotide to bead ratio in the range of about 1:2 to 1:500. In some embodiments, the polynucleotide to bead ratio comprises about 1:2, or about 1:5, or about 1:10, or about 1:25, or about 1:50, or about 1:75, or about 1:100, or about 1:125, or about 1:150, or about 1:175, or about 1:200, or about 1:225, or about 1:250. In some embodiments, the nucleic acid amplification reaction may produce beads that do not have polynucleotides attached thereto, other beads that have one type of polynucleotide attached thereto, and other beads that have more than one type of polynucleotide attached thereto.

In some embodiments, the reaction mixture comprises one or more primers. For example, the reaction mixture may comprise at least a first oligonucleotide primer. In some embodiments, the first primer may comprise a forward amplification primer that hybridizes to at least a portion of one strand of the polynucleotide. In some embodiments, the first primer comprises an extendable 3' terminus for nucleotide polymerization.

In some embodiments, the method for nucleic acid amplification comprises an additional primer hybridized to the template. For example, the second primer may be a reverse amplification primer that hybridizes to at least a portion of one strand of the polynucleotide. In some embodiments, the second primer comprises an extendable 3' terminus. In some embodiments, the second primer is not attached to the surface.

In some embodiments, the third primer may be a forward amplification primer that hybridizes to at least a portion of one strand of the polynucleotide. In some embodiments, the third primer comprises an extendable 3' terminus. In some embodiments, the third primer is not attached to the surface. In some embodiments, the third primer comprises a binding partner or affinity moiety (e.g., biotin) for enriching the amplified nucleic acid.

In some embodiments, the primers (e.g., first, second, and third primers) comprise single stranded oligonucleotides.

In some embodiments, at least a portion of the primer may hybridize to a portion of at least one strand of a polynucleotide in the reaction mixture. For example, at least a portion of the primer may hybridize to a nucleic acid adaptor attached to one or both ends of the polynucleotide. In some embodiments, at least a portion of the primer may be partially or fully complementary to a portion of the polynucleotide or a nucleic acid adaptor. In some embodiments, the primers may be suitable for use in any type of sequencing platform including chemical degradation, chain termination, sequencing by synthesis, pyrophosphate, massively parallel, ion sensitive, and single molecule platforms.

In some embodiments, a primer (e.g., a first, second, or third primer) can have a 5 'or 3' protruding tail (tailed primer) that does not hybridize to a portion of at least one strand of a polynucleotide in a reaction mixture. In some embodiments, the tailed primer may have any length, including a length of 1-50 or more nucleotides.

In some embodiments, the primer comprises a polymer of deoxynucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, the primers comprise naturally occurring, synthetic, recombinant, cloned, amplified, or unamplified forms. In some embodiments, the primers comprise DNA, cDNA, RNA or chimeric RNA/DNA and nucleic acid analogs.

In some embodiments, the primer may be any length, including about 5-10 nucleotides, or about 10-25 nucleotides, or about 25-40 nucleotides, or about 40-55 nucleotides, or longer.

In some embodiments, the method for nucleic acid amplification may include one or more different polymerases. In some embodiments, the polymerase includes any enzyme or fragment or subunit thereof that can catalyze the polymerization of nucleotides and/or nucleotide analogs. In some embodiments, the polymerase requires an extendable 3' terminus. For example, a polymerase requires a terminal 3' OH of a nucleic acid primer to initiate nucleotide polymerization.

The polymerase includes any enzyme that catalyzes the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization may occur in a template-dependent manner. In some embodiments, the polymerase may be a high fidelity polymerase. Such polymerases can include, but are not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase may be a mutant polymerase comprising one or more mutations, including substitution of one or more amino acids with other amino acids, insertion or deletion of one or more amino acids from the polymerase, or ligation of portions of two or more polymerases. As used herein, belonging to "polymerase" and variants thereof also refer to fusion proteins comprising at least two moieties linked to each other, wherein a first moiety comprises a peptide that catalyzes the polymerization of nucleotides into a nucleic acid strand and is linked to a second moiety comprising a second polypeptide, such as a reporter enzyme or sustained synthesis capability-promoting enzyme. Typically, the polymerase comprises one or more active sites on which nucleotide binding and/or catalysis of nucleotide binding can occur. In some embodiments, the polymerase comprises or lacks other enzymatic activities such as 3 'to 5' exonuclease activity or 5 'to 3' exonuclease activity. In some embodiments, the polymerase may be isolated from the cell or produced using recombinant DNA techniques or chemical synthesis methods. In some embodiments, the polymerase may be expressed in a prokaryotic, eukaryotic, viral, or phage organism. In some embodiments, the polymerase may be a post-translationally modified protein or fragment thereof.

In some embodiments, the polymerase may include any one or more of the polymerases or biologically active fragments of the polymerase described in U.S. patent publication No. 2011/0262903 to Davidson et al, 10/27, 2013 and/or international PCT publication No. WO 2013/023176 to Vander Horn et al, 14, 2, 2013.

In some embodiments, the polymerase may be a DNA polymerase and include, but are not limited to, bacterial DNA polymerase, eukaryotic DNA polymerase, archaeal DNA polymerase. Viral DNA polymerase and phage DNA polymerase.

In some embodiments, the polymerase may be a replicase, a DNA-dependent polymerase, a primer enzyme, an RNA-dependent polymerase (including RNA-dependent DNA polymerases such as reverse transcriptase), a thermostable polymerase, or a thermostable polymerase. In some embodiments, the polymerase may be any family a or B type polymerase. Many types of polymerase of families A (e.g., E.coli Pol I), B (e.g., E.coli Pol II), C (e.g., E.coli Pol III), D (e.g., archaebacterium Pol II), X (e.g., human Pol beta), and Y (e.g., E.coli UmuC/DinB and eukaryotic RAD 30/coloring xeroderma lesions) are described in Rothwell and Watsman 2005Advances in Protein Chemistry71:401-440. In some embodiments, the polymerase may be a T3, T5, T7, or SP6RNA polymerase.

In some embodiments, the nucleic acid amplification reaction may be performed using one type or mixture of polymerases, recombinases, and/or ligases. In some embodiments, the nucleic acid amplification reaction may be performed using a low fidelity or high fidelity polymerase.

In some embodiments, the nucleic acid amplification reaction may be catalyzed by a thermostable or thermostable polymerase.

In some embodiments, the polymerase may lack 5'-3' exonuclease activity. In some embodiments, the polymerase may have strand displacement activity.

In some embodiments, the paleodna polymerase may be, but is not limited to, a thermostable or thermophilic DNA polymerase such as: bacillus subtilis (Bacillus subtilis) (Bsu) DNA polymerase I large fragment; thermus aquaticus (Taq) DNA polymerase; a filamentous thermus (Thermus filiformis) (Tfi) DNA polymerase; phi29 DNA polymerase; bacillus stearothermophilus (Bst) DNA polymerase; 9°n-7DNA polymerase of archaea hyperthermophiles (Thermococcus sp.); bacillus smithii (Bsm) DNA polyLarge fragments of synthases; seashore thermophilic cocci (Thermococcus litoralis) (Tli) DNA polymerase or Vent ^TM (exo-) DNA polymerase (from New England Biolabs); or "Deep Vent" (exo-) DNA polymerase (New England Biolabs).

In some embodiments, methods for nucleic acid amplification may include one or more types of nucleotides. Nucleotides include any compound that can selectively bind to or be polymerized by a polymerase. Typically, but not necessarily, selective binding of nucleotides to the polymerase is followed by polymerization of the nucleotides into the nucleic acid strand by the polymerase; occasionally, however, the nucleotides may dissociate from the polymerase without incorporation into the nucleic acid strand, an event referred to herein as a "non-productive" event. Such nucleotides include not only naturally occurring nucleotides but also any analogues (regardless of their structure) that can selectively bind to or be polymerized by a polymerase. Although naturally occurring nucleotides typically comprise base, sugar and phosphate moieties, the nucleotides of the present disclosure may include compounds lacking any, some or all of such moieties. In some embodiments, the nucleotide may optionally comprise a chain of phosphorus atoms comprising 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorus atoms. In some embodiments, the phosphorus chain may be attached to any carbon of the sugar ring, such as the 5' carbon. The phosphorus chain may be attached to the sugar using an inserted O or S. In one embodiment, one or more of the phosphorus atoms in the chain may be part of a phosphate group having P and O. In another embodiment, intervening O, NH, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH may be utilized ₂ 、C(O)、C(CH ₂ )、CH ₂ CH ₂ Or C (OH) CH ₂ R (where R may be 4-pyridine or 1-imidazole) connects together the phosphorus atoms in the chain. In one embodiment, the phosphorus atom in the chain may have a molecular chain containing O, BH ₃ Or a pendant group of S. In the phosphorus chain, the phosphorus atom having a side group other than O may be a substituted phosphoric acid group. In the phosphorus chain, the phosphorus atom having an inserted atom other than O may be a substituted phosphoric acid group. Some examples of nucleotide analogs are described in U.S. patent No. 7,405,281 to Xu.

Some examples of nucleotides that can be used in the disclosed methods and compositions include, but are not limited to, ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, ribonucleotide polyphosphates, deoxyribonucleotide polyphosphates, modified ribonucleotide polyphosphates, modified deoxyribonucleotide polyphosphates, peptide nucleotides, modified peptide nucleotides, metal nucleosides, phosphonate nucleosides, and modified phosphate-sugar backbone nucleotides, analogs, derivatives, or variants of the foregoing, and the like. In some embodiments, the nucleotides may comprise a non-oxygen moiety, such as a sulfur-or borane-moiety, that replaces the oxygen moiety of the alpha phosphate and sugar of a bridging nucleotide, or the alpha and beta phosphate of a nucleotide, or the beta and gamma phosphate of a nucleotide, or between any other two phosphates of a nucleotide, or any combination thereof.

In some embodiments, the nucleotide is unlabeled. In some embodiments, the nucleotide comprises a label and is referred to herein as a "labeled nucleotide". In some embodiments, the label may be in the form of a fluorescent dye attached to any portion of the nucleotide, including the base, sugar, or any phosphate group intervening between them or terminal phosphate group, i.e., the phosphate group furthest from the sugar.

In some embodiments, the nucleotide (or analog thereof) may be linked to a label. In some embodiments, the label comprises an optically detectable moiety. In some embodiments, the tag includes a moiety that is not normally present in a naturally occurring nucleotide. For example, the label may comprise a fluorescent, luminescent or radioactive moiety.

In some embodiments, the method for nucleic acid amplification may further comprise an enrichment step. In some embodiments, the method for nucleic acid amplification can produce at least one bead having attached thereto a plurality of polynucleotides having a sequence complementary to a template polynucleotide (e.g., amplified nucleic acid). At least one of the polynucleotides attached to the bead may hybridize to a polynucleotide having a biotinylated moiety (e.g., a reverse amplification product having a third primer). In some embodiments, the enriching step comprises by combining a polynucleotide having a biotinylated moiety Acid and purified beads (e.g., paramagnetic beads) attached to streptavidin moieties (e.g., myOne from Dynabeads ^TM Bead) are combined to form a purified complex. In some embodiments, the purification complex may be separated/removed from the reaction mixture by utilizing the attractive force of a magnet.

In some embodiments, amplicons comprising a population of substantially monoclonal nucleic acids are each placed, distributed, or positioned at a different site in an array of sites.

In some embodiments, the disclosed methods include dispensing, placing, or otherwise positioning a single template molecule (e.g., a single target polynucleotide of a sample) into a reaction chamber or site (e.g., in an array). A single polynucleotide may be dispensed from a sample into a reaction chamber by flowing a fluid having a polynucleotide sample through the reaction chamber. The individual polynucleotides that are dispensed into the reaction chamber may be single-stranded or double-stranded. In some embodiments, the nucleic acid is amplified in a reaction chamber or site after partitioning.

In some embodiments, different individual target polynucleotides may be dispensed from a sample into each of the different reaction chambers arranged in the array. Different individual polynucleotides may be dispensed from a sample into each of the different reaction chambers by flowing a fluid having a polynucleotide sample through the reaction chambers. The different individual polynucleotides assigned to each of the different reaction chambers may be single-stranded or double-stranded.

In some embodiments, the method comprises partitioning a single polynucleotide into a reaction chamber, and amplifying the single polynucleotide within the reaction chamber, thereby generating a population of monoclonal nucleic acids in the reaction chamber.

In some embodiments, methods for partitioning a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprise a nucleic acid sample. In some embodiments, a single polynucleotide or a different single polynucleotide may be dispensed from a nucleic acid sample having multiple polynucleotides. For example, the nucleic acid sample may comprise from about 2 to about 10, or from about 10 to about 50, or from about 50 to about 100, or from about 100 to about 500, or from about 500 to about 1,000, or from about 1,000 to about 5,000, or about 10 ³ -10 ⁶ Or more than oneDifferent polynucleotides. Different polynucleotides may have the same or different sequences. Different polynucleotides may have the same or different lengths. The sample may comprise single-stranded or double-stranded polynucleotides or a mixture of both.

In some embodiments, the method for partitioning a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprises partitioning a single polynucleotide. In some embodiments, a single polynucleotide may include both single-stranded and double-stranded nucleic acid molecules. In some embodiments, the nucleic acid may include a polymer of deoxynucleotides, ribonucleotides, and/or analogs thereof. In some embodiments, the nucleic acid may comprise a naturally occurring, synthetic, recombinant, cloned, amplified, unamplified, or archived (e.g., preserved) form. In some embodiments, the nucleic acid may include DNA, cDNA, RNA or chimeric RNA/DNA and nucleic acid analogs. In some embodiments, a single polynucleotide may comprise a nucleic acid library construct comprising a nucleic acid linked at one or both ends to an oligonucleotide linker. In some embodiments, the nucleic acid library constructs may be suitable for use in any type of sequencing platform including chemical degradation, chain termination, sequencing by synthesis, pyrophosphate, massively parallel, ion sensitive, and single molecule platforms.

In some embodiments, the sites of the array may include one or more reaction chambers (which may be wells on a solid surface). The reaction chamber may have an aperture defining a width and a depth. The dimensions of the reaction chamber may be sufficient to allow deposition of reagents or for carrying out the reaction. The reaction chamber may have any shape including cylindrical, polygonal, or a combination of different shapes. Any wall of the reaction chamber may have a smooth or irregular surface. The reaction chamber may comprise a bottom having a planar, concave or convex surface. The bottom and side walls of the reaction chamber may comprise the same or different materials and/or may be coated with chemical groups that can react with biomolecules such as nucleic acids, proteins or enzymes.

In some embodiments, the reaction chamber may be a plurality of reaction chambers arranged in a grid or array. The array may comprise two or more reaction chambers. The plurality of reaction chambers may be randomly arranged or arranged in an ordered array. An ordered array may include reaction chambers arranged in rows or in a two-dimensional grid having rows and columns.

The array may include any number of reaction chambers for depositing reagents and performing multiple individual reactions. For example, the array may include at least 256 reaction chambers, or at least 256,000, or at least 1-3 million, or at least 3-5 million, or at least 5-7 million, or at least 7-9 million, at least 9-11 million, at least 11-13 million reaction chambers, or even higher densities including 13-700 million reaction chambers or more. The reaction chambers arranged in a grid may have a center-to-center distance (e.g., pitch) between adjacent reaction chambers of less than about 10 microns, or less than about 5 microns, or less than about 1 micron, or less than about 0.5 microns.

The array may include reaction chambers having any width and depth dimension. For example, the reaction chamber can have a size that accommodates a single particle (e.g., a microbead) or multiple particles. The reaction chamber may contain a water volume of 0.001 to 100 picoliters.

In some embodiments, at least one reaction chamber may be coupled to one or more sensors or may be welded over one or more sensors. The reaction chamber coupled to the sensor may provide a closure of reagents deposited therein so that products from the reaction may be detected by the sensor. The sensor may detect a change in the product from any type of reaction, including any nucleic acid reaction such as primer extension, or nucleotide incorporation reaction. The sensor may detect changes in ions (e.g., hydrogen ions), protons, phosphate groups, such as pyrophosphate groups. In some embodiments, at least one reaction chamber may be coupled to one or more Ion Sensitive Field Effect Transistors (ISFETs). Examples of arrays of reaction chambers coupled to ISFET sensors can be found in U.S. patent No. 7,948,015 and U.S. serial No. 12/002,781.

In some embodiments, the amplification methods (and related compositions, systems, and devices) described herein can be performed in an array of reaction chambers, wherein the reaction chambers of the array form part of a single fluidic system. In some embodiments, the array of multiple reaction chambers may include fluidic interfaces that allow a fluid (e.g., a liquid or gas) to flow through the reaction chambers in a controlled laminar flow. In some embodiments, the array of reaction chambers may include a fluid head space above the reaction chambers for laminar flow. In some embodiments, the array of reaction chambers may be a flow cell or part of a flow chamber, wherein the reaction chambers are in fluid communication with each other. For example, fluid may flow over the array to at least partially or completely fill one or more reaction chambers of the array. In some embodiments, the fluid may completely fill the plurality of reaction chambers and excess fluid may flood the top of the reaction chambers to form a fluid layer over the reaction chambers. The fluid layer above the reaction chamber may provide fluid communication for a plurality of reaction chambers in the array. In some embodiments, fluid communication between multiple reaction chambers in an array may be used to perform separate parallel reactions in the multiple reaction chambers. For example, fluid communication may be used to deliver polynucleotides and/or reagents to multiple reaction chambers to perform parallel nucleic acid amplification reactions.

In some embodiments, a sample having a plurality of different polynucleotides may be applied to a flow chamber to dispense a single polynucleotide to each reaction chamber in an array. In some embodiments, additional reagents may be applied to the flow chamber to dispense into each reaction chamber in the array. For example, additional reagents may include microparticles, one or more enzymes, enzyme cofactors, primers, and/or nucleoside triphosphates. In some embodiments, polynucleotides and reagents may be delivered to the array of reaction chambers in any order, including sequentially or substantially simultaneously or a combination of both. For example, in some embodiments, polynucleotides may be dispensed first to an array of reaction chambers followed by dispensing reagents, or the reverse order may be used, or polynucleotides and reagents may be dispensed substantially simultaneously.

In some embodiments, any method (including flow cells) may be used to deliver polynucleotides and/or reagents to a percentage of the reaction chambers in an array. For example, the array of loaded reaction chamber percentage including about 1-25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or higher percentage. In some embodiments, the percentage of reaction chambers loaded with polynucleotides and/or reagents may be increased by performing two or more rounds of loading steps. For example, (a) in a first round, polynucleotides and/or reagents may be dispensed to multiple reaction chambers in an array, and (b) in a second round, polynucleotides and/or reagents may be dispensed to the same array. Additional loading rounds (e.g., third, fourth, or more rounds) may be performed. In some embodiments, any type of reaction may be performed between any loading rounds and/or may be performed after completion of multiple loading rounds. For example, the nucleic acid amplification reaction may be performed between any loading rounds or may be performed after completion of a plurality of loading rounds. In some embodiments, after each loading round, compounds can be layered on the array to prevent polynucleotides and beads from migrating out of the reaction chamber. For example, after each loading round, a solution comprising at least one sieving agent may be layered on the array. In some embodiments, the sieving agent comprises a cellulose derivative. Alternatively, an oil layer may be layered over the reaction mixture in the wells or chambers of the array.

In some embodiments, the disclosed methods (and related compositions, systems, and kits) further comprise ligating the nucleic acid template to the sites of the array prior to amplifying the template. Optionally, the site comprises a primer, and the ligating comprises hybridizing the primer to a primer binding site of the template. For example, a site within the array may comprise at least one immobilized primer comprising a sequence that is at least partially complementary to a primer binding site of a template that is assigned or placed at the site. The primers facilitate ligation of the templates to the array. In some embodiments, a majority of the sites in the array comprise at least one primer. The primers at different sites may be identical to each other. Alternatively, the primers at different sites may be different from each other. In an exemplary embodiment, at least two sites each comprise a different target-specific primer.

The primers may be attached to the sites of the array using any suitable method. Microparticles attach primers to the surface of a nano-array of reaction chambers (e.g., ISFET array type for ion-based sequencing), it may be useful to first synthesize or prepare a three-dimensional matrix within at least some of the reaction chambers of the array. In embodiments, the polymer matrix precursor may be applied to an array of wells associated with one or more sensors. The polymer matrix precursors can be polymerized to form an array of polymer matrices. These polymer matrices can be conjugated to oligonucleotides and can be used in a variety of analytical techniques including genetic sequencing techniques.

In some embodiments, the hydrophilic polymer matrix is dispensed in wells associated with a sensor (e.g., a sensor of a sensor array). In an example, the hydrophilic matrix is a hydrogel matrix. The hydrophilic matrix may correspond to the sensors of the sensor array in a one-to-one configuration. In other examples, the sensors of the sensor array may include Field Effect Transistor (FET) sensors, such as Ion Sensitive Field Effect Transistors (ISFETs). In particular, the matrix material is cured in situ and is adapted to the structure of the individual pores of the sensing device. The void areas between the pores may be substantially free of polymer matrix. In an example, the matrix material may be bonded, e.g., covalently bonded, to the surface of the pores. In an example, the holes have a depth or thickness in the range of 100 nanometers to 10 microns. In another example, the pores may have a characteristic diameter in the range of 0.1 microns to 2 microns.

In an exemplary method, the polymer matrix can be formed by applying an aqueous solution comprising a polymer precursor to the wells of the array of wells. The volume of aqueous material defined by the array of wells may be isolated using an immiscible fluid disposed over the array of wells. The isolated volume of solution may be initiated to promote polymerization of the matrix precursor resulting in an array of matrices dispensed within the well. In an example, an aqueous solution comprising a matrix precursor is dispensed to the pores of the sensing device by flowing the aqueous precursor through the pores. In another example, the aqueous solution is included as a dispersed phase in an emulsion. The dispersed phase may settle or be excited in the pores of the pore array. The polymerization of the matrix precursor may be initiated using an initiation factor placed in the aqueous phase or in an immiscible fluid. In another example, the polymerization may be initiated thermally.

In another exemplary method, a matrix array can be formed within the wells of a sensing device by anchoring starting molecules at a surface within the wells of the well array. A solution comprising a matrix precursor may be provided on the wells of the well array. The initiation factor may initiate polymerization of the matrix precursor resulting in the formation of a polymer matrix within the pores of the pore array. In other examples, aspects of the above methods are combined to further enhance the formation of the matrix array.

In particular embodiments, the sequencing system includes a flow cell in which the sensor array is disposed, a communication circuit in electronic communication with the sensor array, and a container in fluid communication with the flow cell and a fluid control. In an example, fig. 13 shows an expanded view and a cross-sectional view of the flow cell 100 and shows a portion of the flow chamber 106. The reagent flow 108 flows across the surface of the array of wells 102, wherein the reagent flow 108 flows over the open ends of the wells of the array of wells 102. The array of apertures 102 and the array of sensors 105 together may form an integrated unit that forms the lower wall (or bottom surface) of the flow cell 100. The reference electrode 104 can be fluidly coupled to the flow chamber 106. In addition, the flow cell cover 130 encloses the flow chamber 106 to contain the reagent flow 108 within a confined area.

Fig. 14 shows an expanded view of aperture 201 and sensor 214 (as shown at 110 of fig. 13). The pore volume, shape, aspect ratio (e.g., bottom width to pore depth ratio) and other dimensional characteristics may be selected based on the nature of the reaction that occurs and the labeling technique (if any) or reagents, byproducts used. Sensor 214 may be a chemical field effect transistor (chemFET), more particularly an Ion Sensitive FET (ISFET), having a floating gate 218, the floating gate 218 having a sensor plate 220 that is optionally separated from the interior of the well by a layer of material 216. In addition, a conductive layer (not shown) may be placed on the sensor board 220. In an example, the material layer 216 includes an ion-sensitive material layer. The material layer 216 may be a ceramic layer such as an oxide of zirconium, hafnium, tantalum, aluminum or titanium or a nitride of titanium, among others. In examples, the material layer 216 may have a thickness of 5nm to 100nm, such as 10nm to 70nm,15nm to 65nm, or 20nm to 50 nm.

Although material layer 216 is shown as extending beyond the boundaries of the FET assembly shown, material layer 216 may extend along the bottom of aperture 201 and optionally along the walls of aperture 201. The sensor 214 may sense (and generate an associated output signal) the amount of charge 224 present on the material layer 216 opposite the sensor plate 220. The change in charge 224 may cause a change in current between the source 221 and drain 222 of the chemFET. Further, chemfets may be used directly to provide a current-based output signal or indirectly with additional circuitry to provide a voltage-based output signal. Reactants, wash solutions, and other reagents may move into and out of the pores through diffusion mechanism 240.

In embodiments, the reaction performed in well 201 may be an analytical reaction to identify or determine a characteristic or property of an analyte of interest. Such a reaction may directly or indirectly produce byproducts that affect the amount of charge near the sensor plate 220. If such byproducts are generated in small amounts or decay rapidly or react with other components, multiple copies of the same analyte can be simultaneously analyzed in well 201 to increase the output signal generated. In embodiments, multiple copies of the analyte may be attached to the solid support 212 either before or after placement into the well 201. The solid support 212 may be a polymer matrix, such as a hydrophilic polymer matrix, such as a hydrogel matrix or the like. For simplicity and ease of description, the solid support 212 is also referred to herein as a polymer matrix.

The aperture 201 may be defined by a wall structure, which may be formed from one or more layers of material. In examples, the wall structure may have a thickness extending from the lower surface to the upper surface of the hole of 0.01 microns to 10 microns, such as 0.05 microns to 10 microns, 0.1 microns to 10 microns, 0.3 microns to 10 microns, or 0.5 microns to 6 microns. In particular, the thickness may be in the range of 0.01 microns to 1 micron, for example in the range of 0.05 microns to 0.5 microns, or 0.05 microns to 0.3 microns. The holes 201 may have a characteristic diameter of no more than 5 microns, such as no more than 3.5 microns, no more than 2.0 microns, no more than 1.6 microns, no more than 1.0 microns, no more than 0.8 microns, or no more than 0.6 microns, defined by 4 times the cross-sectional area (a) divided by the square root of Pi (e.g., sqrt (4*A/Pi)). In an example, the holes 201 may have a characteristic diameter of at least 0.01 microns.

Although fig. 14 shows a single wall structure and a single layer of material 216, the system may include one or more wall structure layers, one or more conductive layers, or one or more layers of material. For example, the wall structure may be formed from one or more layers comprising TEOS or an oxide of silicon or comprising a nitride of silicon.

In the specific example shown in fig. 15, the system 300 includes a hole wall structure 302 defining an array of holes 304 disposed on or operatively coupled to a sensor pad of a sensor array. The cell wall structure 302 defines an upper surface 306. The lower surface 308 associated with the aperture rests on the sensor pad of the sensor array. The aperture wall structure 302 defines a sidewall 310 between the upper surface 306 and the lower surface 308. As described above, the layer of material in contact with the sensor pads of the sensor array may extend along the lower surface 308 of the wells of the array of wells 304 or along at least a portion of the walls 310 defined by the well wall structures 302. The upper surface 306 may be free of a layer of material. In particular, the polymer matrix may be disposed in the wells of the array of wells 304. The upper surface 306 may be substantially free of polymer matrix. For example, the upper surface 306 may include an area without the polymer matrix, such as at least 70% of the total area, at least 80% of the total area, at least 90% of the total area, or about 100% of the total area.

Although the wall surface of fig. 14 is shown as extending substantially perpendicularly and outwardly, the wall surface may extend in different directions and may have different shapes. The term "substantially perpendicular" means extending in a direction of a component orthogonal to the surface defined by the sensor pad. For example, as shown in fig. 16, the aperture wall 402 may extend vertically, parallel to a normal component 412 of the surface defined by the sensor pad. In another example, the wall surface 404 extends substantially perpendicularly in an outward direction from the sensor pad, providing a larger aperture opening than the lower surface area of the aperture. As shown in fig. 16, wall surface 404 extends in a perpendicular component direction parallel to normal component 412 of surface 414. In an alternative example, the wall surface 406 extends substantially vertically in an inward direction, providing an opening area that is smaller than the lower surface area of the aperture. Wall surface 406 extends in a component direction parallel to normal component 412 of surface 414.

Although

surfaces

402, 404, or 406 are shown by straight lines, some semiconductor or CMOS fabrication processes may result in structures having non-linear shapes. In particular, wall surfaces, such as wall surface 408, and upper surfaces, such as upper surface 410, may be arcuate in shape or may have various non-linear forms. Although the structures and devices shown along are described as having linear layers, surfaces or shapes, the actual layers, surfaces or shapes produced by the semiconductor process may differ to some extent, possibly including non-linear and arcuate variations of the embodiments shown.

Fig. 17 shows an exemplary aperture containing a layer of ion sensitive material. For example, the aperture structure 502 may define an array of apertures, such as

exemplary apertures

504, 506, or 508. The aperture (504, 506 or 508) may be operatively coupled to an underlying sensor (not shown) or connected to such an underlying sensor. The exemplary aperture 504 includes a layer 510 of ion sensitive material defining the bottom of the aperture 504 and extending into the structure 502. Although not shown in fig. 17, a conductive layer, such as a gate, for example, a floating gate of an ion sensitive field effect transistor, may be located below the ion sensitive material layer 510.

In another example, as shown by the holes 506, the ion sensitive material layer 512 may define the bottoms of the holes 506 without extending into the structure 502. In other examples, the aperture 508 may include an ion sensitive layer 514 extending along at least a portion of a sidewall 516 of the aperture 508 defined by the structure 502. As above, the ion

sensitive material layer

512 or 514 may be located over an underlying conductive layer or gate of an electronic device.

In fig. 14, the matrix material 212 is conformal to the pore structure. In particular, the matrix material may be cured in situ to conform to the walls and floor of the hole. The upper surface defining the aperture may comprise an area substantially free of matrix material, such as at least 70% of the total area, at least 80% of the total area, at least 90% of the total area, or about 100% of the total area. Depending on the nature of the pore structure, the polymer matrix may be physically fixed to the pore wall structure. In another example, the polymer matrix may be chemically bonded to the pore wall structure. In particular, the polymer matrix may be covalently bound to the pore wall structure. In another example, the polymer matrix may be bonded to the pore wall structure by hydrogen bonding or ionic bonding.

The polymer matrix may be formed from matrix precursors, such as monomers that are fully polymerizable, such as vinyl-based monomers. Specifically, the monomer may include hydrophilic monomers such as acrylamide, vinyl acetate, hydroxyalkyl methacrylate, variants or derivatives thereof, copolymers thereof, or any combination thereof. In a specific example, the hydrophilic monomer is an acrylamide, such as an acrylamide functionalized to include hydroxyl groups, amino groups, carboxyl groups, halo groups, or combinations thereof. In examples, the hydrophilic monomer is an aminoalkylacrylamide, an acrylamide functionalized with an amine-terminated polyacryl alcohol (D, shown below), an propenpiperazine (C, shown below), or a combination thereof. In another example, the acrylamide may be a hydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. In particular, the hydroxyalkyl acrylamide may include N-tris (hydroxymethyl) methyl) acrylamide (a, shown below), N- (hydroxymethyl) acrylamide (B, shown below), or a combination thereof. In another example, the comonomer may comprise a halogen modified acrylate or acrylamide, such as N- (5-bromoacetamidopentyl) acrylamide (BRAPA, E, shown below). In another example, the comonomer may comprise an oligonucleotide modified acrylate or acrylamide monomer. In other examples, a mixture of monomers may be used, such as a mixture of hydroxyalkyl acrylamide and amine functionalized acrylamide or a mixture of acrylamide and amine functionalized acrylamide. In examples, the amine-functionalized acrylamide may be included in a ratio of hydroxyalkyl acrylamide to amine-functionalized acrylamide or acrylamide to amine-functionalized acrylamide in a range of 100:1 to 1:1, such as in a range of 100:1 to 2:1, 50:1 to 3:1, 50:1 to 5:1, or 50:1 to 10:1. In another example, the amine-functionalized acrylamide may be included in a ratio of hydroxyalkyl acrylamide to bromo-functionalized acrylamide or acrylamide to bromo-functionalized acrylamide in a range of 100:1 to 1:1, such as in a range of 100:1 to 2:1, 50:1 to 3:1, 50:1 to 5:1, or 50:1 to 10:1.

In other examples, oligonucleotide-functionalized acrylamide or acrylate monomers, e.g., acrydite ^TM Monomers to incorporate the oligonucleotides into a polymer matrix.

Another exemplary matrix precursor includes a cross-linking agent. In examples, the crosslinker is included in a mass ratio of monomer to crosslinker of 15:1 to 1:2, e.g., 10:1 to 1:1, 6:1 to 1:1, or 4:1 to 1:1. In particular, the cross-linking agent may be a divinyl cross-linking agent. For example, the divinyl crosslinker may comprise bis-acrylamide, such as N, N '- (ethane-1, 2-diyl) bis (2-hydroxyethyl) acrylamide, N' - (2-hydroxypropane-1, 3-diyl) bis-acrylamide, or a combination thereof. In another example, the divinyl crosslinker comprises ethylene glycol dimethacrylate, divinylbenzene, cyclohexane bisacrylamide, trimethylol propane trimethacrylate, protected derivatives thereof, or combinations thereof.

In one aspect, the polymer network comprises a polyacrylamide gel having a total monomer percentage in the range of 3-20%, more preferably in the range of 5-10%. In one embodiment, the percentage of crosslinker of the monomer is in the range of 5-10%. In a specific embodiment, the polymer network comprises 10% total acrylamide, wherein 10% is a bisacrylamide crosslinker.

Polymerization may be initiated by an initiation factor in solution. For example, the initiation factor may be water-based. In another example, the initiation factor may be a hydrophobic initiation factor, preferably located in a hydrophobic phase. Exemplary initiation factors include ammonium persulfate or TEMED (tetramethyl ethylenediamine). TEMED may accelerate the rate of formation of free radicals from persulfate, thereby catalyzing polymerization. Persulfate radicals, for example, convert acrylamide monomers to radicals, which react with unactivated monomers to initiate polymerization chain reactions. The extended polymer chains can randomly crosslink, resulting in a gel with a characteristic porosity that depends on polymerization conditions and monomer concentration. Riboflavin (or riboflavin-5' -phosphate) may also be used as a source of free radicals, which is typically used in combination with TEMED and ammonium persulfate. In the presence of light and oxygen, riboflavin is converted into its colorless form, which has the activity of initiating polymerization, which is commonly referred to as photopolymerization.

In another example, a nitrogen-containing initiation factor may be used to initiate polymerization. Exemplary water-soluble nitrogen-containing start factors are shown in table 1 and exemplary oil-soluble nitrogen-containing start factors are shown in table 2. In particular, the nitrogen-containing initiation factor may be Azobisisobutyronitrile (AIBN).

TABLE I

Water-soluble nitrogenous initiator compounds

Table II

Oil-soluble nitrogenous initiator compounds

In other examples, the precursor of the polymer matrix may include a surface active additive to enhance binding to the surface. Exemplary additives include functionalized acrylic monomers or functionalized acrylamide monomers. For example, the acrylic monomer may be functionalized to bind surface materials, such as ceramic materials that form the bottom or sidewalls of the pores. In an example, the additive may include a propenyl-phosphonate, such as a methacrylic phosphonate. In another example, the additive may include dimethylacrylamide or polydimethylacrylamide. In other examples, the additive may include polylysine modified with polymerizable groups (e.g., acrylic groups).

In another example, atom Transfer Radical Polymerization (ATRP) may be used to facilitate polymerization. ATRP systems may include a starter factor, a transition metal ion, and a ligand. Exemplary transition metal ion complexes include copper-based complexes. Exemplary ligands include 2,2 '-bipyridine, 4' -di-5-nonylalkyl-2, 2 '-bipyridine, 4',4 "-tris (5-nonylalkyl) -2,2':6',2" -terpyridine, N, N, N ', N', N "-pentamethyldiethylenetriamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, tris (2-dimethylaminoethyl) amine, N, N-bis (2-picolylmethyl) octadecylamine, N, N, N ', N' -tetrakis [ (2-pyridine) methyl ] ethylenediamine, tris [ 2-pyridine) methyl ] amine, tris (2-aminoethyl) amine, tris (2-bis (3-butoxy-3-oxypropyl) aminoethyl) amine, tris (2-bis (3- (2-ethylhexyloxy) -3-oxypropyl) aminoethyl) amine, tris (2-bis (3-dodecyloxy-3-oxypropyl) aminoethyl) amine, variants and derivatives thereof, or combinations thereof. Exemplary initiation factors include 2-bromopropionitrile, ethyl 2-bromoisobutyrate, ethyl 2-bromopropionate, methyl 2-bromopropionate, 1-phenylbromoethane, p-toluenesulfonyl chloride, 1-cyano-1-methylethyldiethyl dithiocarbamate, ethyl 2- (N, N-diethyldithiocarbamate) -isobutyrate, dimethyl 2, 6-dibromoheptanedionate, variants or derivatives thereof, and any combination thereof. Optionally, BRAPA monomers can be used as branching agents in the presence of ATRP systems.

In an example, ATRP is initiated at the surface to directly bond the polymer to the surface. For example, an acrylic monomer, an acrylamide monomer, acrydite may be used in the presence of a transition metal ion/ligand complex ^TM Monomers, succinimidyl acrylates, diacrylic or bisacrylamide monomers, derivatives thereof, or combinations thereof are applied to the starting surface in solution.

In another, ATRP systems can be used to attach polymers to the surface of the pores using modified phosphonate, sulfonate, silicate or zirconate compounds. In particular, amine or hydroxyl terminated alkyl phosphonates or alkoxy derivatives thereof may be applied to a surface and initiated using a initiation factor. The catalyst complex and monomer may be applied to extend the surface compound.

In an exemplary method, an aqueous solution comprising a precursor of a polymer matrix may be applied into the pores of a structure defining an array of pores. The aqueous solution in the well may be isolated by providing a non-miscible fluid over the well and initiating polymerization of the polymer precursor in solution within the well.

For example, FIG. 18 shows an exemplary pore structure 602 defining pores 604. One or more sensors (not shown) may be operably coupled or connected to the aperture 604. For example, one or more sensors may include a gate structure in electrical communication with at least a bottom surface of the aperture 604. An aqueous solution 606 containing a polymer precursor among other components is provided over the pores and a solution containing the polymer precursor is dispensed into the pores 604. Exemplary polymer precursors include, among others, monomers, crosslinkers, initiation factors, or surfactants, e.g., as described above. Optionally, the pores 604 may be wetted prior to deposition using a hydrophilic solution, such as a solution comprising water, an alcohol, or a mixture thereof, or a solution comprising water and a surfactant. Exemplary alcohols include isopropanol. In another example, the alcohol comprises ethanol. Although not shown, the bottom surface of the well and optionally the sidewalls of the well may comprise an ion sensitive material. Such ion sensitive materials may overlie the conductive structures of underlying electronic devices such as field effect transistors. One or more surfaces of the pores may be treated with a surface-active additive prior to application of the solution comprising the polymer precursor.

The distribution of the aqueous solution containing the polymer precursor into the pores 604 may be further enhanced by the shaking structure (e.g., by rotation or vortexing). In another example, vibration, such as sonic or ultrasonic vibration, may be used to increase the distribution of the aqueous solution within the pores 604. In other examples, a vacuum pump may be used to degas the wells and apply the solution at a negative gauge pressure. In an example, an aqueous solution is dispensed to the wells at room temperature. In another example, the aqueous solution is dispensed at a temperature below room temperature, particularly when a water-based initiation factor is used. Alternatively, the aqueous solution is dispensed at an elevated temperature.

As shown in fig. 19, an immiscible fluid 708 is applied over the well 604, pushing the aqueous solution 606 off the top of the well and isolating the aqueous solution 606 within the well 604, as shown in fig. 20. Exemplary immiscible fluids include mineral oils, silicone oils (e.g., poly (dimethylsiloxane)), heptanes, carbonate oils (e.g., diethylhexyl carbonate (Tegosoft)

) Or a combination thereof).

The initiation factor may be administered within the aqueous solution 606. Alternatively, the initiation factor may be provided within the immiscible fluid 708. Polymerization may be initiated by changing the temperature of the substrate. Alternatively, the polymerization may occur at room temperature. In particular, the polymer precursor solution may be maintained at a temperature of 20 ℃ to 100 ℃, e.g., 25 ℃ to 90 ℃, 25 ℃ to 50 ℃, or 25 ℃ to 45 ℃ for 10 minutes to 5 hours, e.g., 10 minutes to 2 hours or 10 minutes to 1 hour.

Due to polymerization, an array of polymer matrices 912 is formed within the pores 604 defined by the pore structure 602, as shown in fig. 21. Optionally, the array may be washed with NaOH (e.g., 1N NaOH) to remove polymer from the interstitial regions between the wells.

In an alternative example, an emulsion comprising an aqueous solution of a polymer precursor as a dispersed phase in an immiscible fluid is used to deposit droplets of the aqueous solution within the pores. For example, as shown in fig. 22, the aperture structure 1002 defines an aperture 1004. The aperture may be operatively coupled or electrically connected to one or more sensors (not shown). As described above, the bottom and optionally side surfaces of the walls of the holes 1004 may be defined by a layer of ion-sensitive material that may overlie the conductive components of the underlying electronic device.

The emulsion 1006 may comprise aqueous droplets 1008 comprising a polymer precursor dispersed within a continuous immiscible fluid 1010. The droplet 1008 may settle in the aperture 1004. In particular, aqueous droplets 1008 have a greater density than immiscible fluid 1010. Exemplary immiscible fluids include mineral oil, silicone oil (e.g., poly (dimethylsiloxane)) heptane, carbonate oil (e.g., diethylhexyl carbonate (Tegosoft)

) Or a combination thereof). In other examples, the distribution of the aqueous droplets into the wells 1004 may be facilitated by rotating, vortexing, or sonicating a fluid or structure. Optionally, a hydrophilic solution, such as a solution comprising water, alcohol, or mixtures thereof, or comprising water and a surface active agent, may be used prior to depositionA solution of a sex agent to wet the pores 604. The temperature during dispensing of the droplets into the wells may be at room temperature. Alternatively, the dispensing may be performed at an elevated temperature.

As shown in fig. 23, the droplets combine within the wells 1004 to provide an isolated solution comprising the polymer precursor 1112. Optionally, the emulsion 1006 may be replaced with an immiscible fluid, such as an immiscible fluid 1010 that does not contain droplets 1008 or a different immiscible fluid 1116. Exemplary immiscible fluids include mineral oil, silicone oil (e.g., poly (dimethylsiloxane)) heptane, carbonate oil (e.g., diethylhexyl carbonate (Tegosoft)

) Or a combination thereof). Alternatively, the emulsion 1006 may remain in place during polymerization. In this manner, the solution 1112 within the well 1004 is isolated from the solutions in the other wells 1004. Polymerization may be initiated resulting in a polymer matrix 1214 within the pores 1004, as shown in fig. 24. As described above, the polymerization may be initiated thermally. In another example, an oil phase initiator may be used to initiate polymerization. Alternatively, the polymerization may be initiated using a water phase initiation factor. In particular, a second emulsion may be applied over the aperture 1004. The second emulsion may include a dispersed aqueous phase including an aqueous phase initiation factor.

Polymerization may be initiated by changing the temperature of the substrate. Alternatively, the polymerization may occur at room temperature. In particular, the polymer precursor solution may be maintained at a temperature of 20 ℃ to 100 ℃, e.g., 25 ℃ to 90 ℃, 25 ℃ to 50 ℃, or 25 ℃ to 45 ℃ for 10 minutes to 5 hours, e.g., 10 minutes to 2 hours or 10 minutes to 1 hour. Optionally, the array may be washed with NaOH (e.g., 1 NNaOH) to remove polymer from the interstitial regions between the wells.

In another example, an array of matrix material may be formed within the wells of the well array using an initiation factor immobilized at the surface of the well array. For example, as shown in fig. 25, structure 1302 may define an aperture 1304. The material layer 1306 may define a lower surface of the hole 1304. An anchor compound 1308, such as an anchor compound for Atom Transfer Radical Polymerization (ATRP), may be immobilized to the layer of material 1306 defining the bottom surface of the hole 1304. Alternatively, the sidewall material defined within the hole or layer of structure 1302 exposed within hole 1304 may anchor a compound such as the compound for ATRP described above.

In such examples, a solution 1310 comprising a polymer precursor, such as a monomer, a cross-linking agent, and optionally a surfactant additive, may be applied over the structure 1302 and within the pores 1304. Anchoring compound 1308 can be initiated to promote polymerization extending from the anchoring compound, sequester polymerization within pores 1304, and fix the polymer to pores 1304. In an example, the anchoring compound has a surface active group and a distal radical forming group. The surface active groups may include phosphonates, silicate sulfonates, zirconates, titanates, or combinations thereof. The distal radical forming group may comprise an amine or hydroxyl group that may undergo transfer with, for example, a halogenated (e.g., brominated) compound and subsequently form radicals for polymerizing the polymer precursor and anchoring the resulting polymer to the pore surface. Typically, ATRP systems are selected to terminate polymerization after a statistically average length or amount of monomer is added. In this manner, the total number of aggregations within the aperture 1304 may be controlled. In other examples, other actual applications and additions to the aqueous solution 1310 that affect chain extension or termination may be made.

In another example shown in fig. 26, structure 1402 may define holes 1404. The aperture 1404 may include a layer of material 1406 extending along the sidewalls 1410 of the structure 1402 and the aperture 1404. Initiator 1408 may be secured to material layer 1406 along the bottom of aperture 1404 and along sidewall 1410. A solution 1412 comprising a polymer precursor, a cross-linking agent, and other agents may be applied over the structure 1402 and the holes 1404.

As shown in fig. 27, the polymer matrix 1512 is formed as a result of an initial polymerization extending from a surface within the aperture 1304 (e.g., a surface defined by the material layer 1306).

Polymerization may be initiated by changing the temperature of the substrate. Alternatively, the polymerization may occur at room temperature. In particular, the polymer precursor solution may be maintained at a temperature of 20 ℃ to 100 ℃, e.g., 25 ℃ to 90 ℃, 25 ℃ to 50 ℃, or 25 ℃ to 45 ℃ for 10 minutes to 5 hours, e.g., 10 minutes to 2 hours or 10 minutes to 1 hour.

After formation, the polymer matrix may be activated to facilitate conjugation to a target analyte, such as a polynucleotide. For example, functional groups on the polymer matrix may be enhanced to allow binding to a target analyte or analyte receptor (e.g., an oligonucleotide primer). In particular examples, the functional groups of the hydrophilic polymer matrix may be modified with an agent capable of converting the hydrophilic polymer functional groups into active moieties that may undergo nucleophilic or electrophilic substitution. For example, hydroxyl groups on the polymer matrix may be activated by substitution of at least a portion of the hydroxyl groups with sulfonic acid groups or chlorine. Exemplary sulfonic acid groups may be derived from trifluoroethane sulfonyl (tresyl), methanesulfonyl, p-toluenesulfonyl, or p-fluorobenzenesulfonyl (fosyl) chloride, or any combination thereof. The sulfonate is used to allow nucleophilic groups to replace the sulfonate. The sulfonate ester may also react with the released chlorine to provide chlorinated functional groups that may be used in the process of conjugating the matrix. In another example, amine groups on the polymer matrix may be activated.

For example, the target analyte or analyte receptor may be bound to the hydrophilic polymer by nucleophilic substitution with a sulfonic acid group. In particular examples, target analyte receptors that are end-capped with a nucleophilic group, such as an amine or sulfhydryl group, may undergo nucleophilic substitution to replace sulfonic acid groups in the polymer matrix. Due to activation, a conjugated polymer matrix may be formed.

In another example, the sulfonated polymer matrix may be further reacted with a mono-or polyfunctional mono-or poly-nucleophile (e.g., maleimide) that can form a linkage to the matrix while preserving the nucleophilic activity for the electrophilic group-containing oligonucleotide. Furthermore, the remaining nucleophilic activity can be converted to electrophilic activity by attachment to a reagent comprising a multiple electrophilic group, which in turn will attach to an oligonucleotide comprising a nucleophilic group.

In another example, the monomer comprising the functional group may be added during polymerization. Monomers include, for example, acrylamides containing carboxylic acid, ester, halogen, or other amine reactive groups. The ester groups may be hydrolyzed prior to reaction with the oligoamines (amine oligomers).

Other conjugation techniques include the use of amine-containing monomers. Amine groups are nucleophilic groups that can be further modified by amine-reactive difunctional amphiphilic electrophiles that produce monofunctional electrophilic groups upon attachment to a polymer matrix. Such electrophilic groups can react with oligonucleotides having nucleophilic groups (e.g., amine or sulfhydryl groups) resulting in ligation of the oligonucleotides (by reaction with the vacated electrophile).

If the polymer matrix is prepared from a combination of amino-and hydroxy-acrylamides, the polymer matrix comprises a combination of nucleophilic amino groups and neutral hydroxyl groups. The amino groups can be modified with a difunctional amphiphilic moiety such as a diisocyanate or di-NHS ester to produce a hydrophilic polymer matrix that is reactive with nucleophilic groups. Exemplary di-NHS esters include di-succinimidyl C2-C12 alkyl esters, such as di-succinimidyl suberate or di-succinimidyl glutarate.

Other activation chemistries include the incorporation of multiple steps to convert specific functional groups to accommodate specific desired linkages. For example, sulfonate modified hydroxyl groups can be converted into nucleophilic groups by several methods. In an example, the reaction of the sulfonate with the azide anion produces an azide-substituted hydrophilic polymer. Azide can be used directly to conjugate to acetylene substituted biomolecules by "CLICK" chemistry, which can be performed with or without copper catalysis. Optionally, the azide may be converted to the amine by, for example, catalytic reduction with hydrogen or reduction with an organophosphine. The resulting amine can then be converted to an electrophilic group using a variety of reagents such as diisocyanate, di-NHS ester, cyanuric chloride, or combinations thereof. In an example, diisocyanates are used to create urea linkages between the polymer and the linker, which results in residual isocyanate groups that are capable of reacting with amino-substituted biomolecules to create urea linkages between the linker and the biomolecules. In another example, the use of a di-NHS ester produces an amide bond between the polymer and the linker and a remaining NHS ester group that is capable of reacting with an amino-substituted biomolecule to produce an amide linkage between the linker and the biomolecule. In other examples, the use of cyanuric chloride creates an amino-triazine bond between the polymer and the linker and two remaining chlorotriazine groups, one of which is capable of reacting with an amino-substituted biomolecule to create an amino-triazine bond between the linker and the biomolecule. Other nucleophilic groups can be incorporated into the matrix by sulfonyl activation. For example, reaction of a sulfonic acid acyl substrate with a thiobenzoic acid anion and hydrolysis of the resulting thiobenzoate incorporates a thiol group into the substrate, which can then be reacted with a maleimide substituted biomolecule to produce a thio-succinimide linkage with the biomolecule.

Mercapto groups may also be reacted with bromo-acetyl or bromo-amidyl groups (bromoo-amidyl groups). In a specific example, when n- (5-bromoacetamidopentyl) acrylamide (BRAPA) is included as a comonomer, the oligonucleotide can be incorporated by forming a thiobenzamide-oligonucleotide compound for reaction with bromo-acetyl groups on the polymer, for example as shown below.

The thiobenzamide-oligonucleotide compound may be formed by reacting the following dithiobenzoic acid-NHS compound with an amine-terminated oligonucleotide and activating the dithiobenzamide-oligonucleotide compound to form the thiobenzamide-oligonucleotide compound shown above.

Alternatively, acrydite oligonucleotides may be used to incorporate the oligonucleotides during polymerization. Exemplary acrydite oligonucleotides may include ion exchanged oligonucleotides.

Covalent attachment of biomolecules to refractory or polymeric substrates can be produced by using electrophilic moieties on the substrate coupled to nucleophilic moieties on the biomolecules or nucleophilic linkages on the substrate coupled to electrophilic linkages on the biomolecules. The solvent selected for these couplings is water or water containing some water-soluble organic solvent to disperse the biomolecules on the substrate due to the hydrophilic nature of most common biomolecules of interest. In particular, polynucleotides are typically coupled to a substrate in an aqueous system because of their polyanionic nature. Since water competes with the nucleophilic group for electrophiles by hydrolyzing the electrophile into a moiety that is inactive for conjugation, aqueous systems can result in low yields of coupled product, where the yield is based on the conjugated electrophilic moiety. When high yields of electrophilic moieties of the reaction coupling body are desired, high concentrations of nucleophilic groups drive the reaction and slow down hydrolysis, resulting in inefficient use of nucleophilic groups. In the case of polynucleic acids, the metal counter ion of the phosphate can be replaced by a lipophilic counter ion to aid in dissolving the biomolecule in a polar, non-reactive, non-aqueous solvent. These solvents may include amides or ureas such as formamide, N-dimethylformamide, acetamide, N, N-dimethylacetamide, hexamethylphosphoric acid amide, pyrrolidone, N-methylpyrrolidone, N ' -tetramethylurea, N ' -dimethyl-N, N ' -trimethylene urea, or combinations thereof; carbonates such as dimethyl carbonate, propylene carbonate, or combinations thereof; esters such as tetrahydrofuran; sulfoxides and sulfones such as dimethyl sulfoxide, dimethyl sulfone, or combinations thereof; hindered alcohols (hindered alcohol) such as t-butyl alcohol; or a combination thereof. Lipophilic cations may include tetraalkylammonium or tetraarylammonium cations such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, tetrapentylammonium, tetrahexylammonium, tetraheptylammonium, tetraoctylammonium, and alkyl and aryl mixtures thereof, tetraarylphosphine cations such as tetraphenylphosphine, tetraalkylarsones or tetraarylarsines such as tetraphenylarsine, and trialkylsulfonium cations such as trimethylsulfonium, or combinations thereof. The conversion of polynucleic acids to organic solvent soluble materials (by exchanging metal cations with lipophilic cations) can be carried out by a variety of standard cation exchange techniques.

In particular embodiments, the polymer matrix is exposed to a target polynucleotide having a segment complementary to an oligonucleotide conjugated to the polymer matrix. The polynucleotide is subjected to amplification, for example by Polymerase Chain Reaction (PCR) or Recombinase Polymerase Amplification (RPA). For example, the target polynucleotides are provided in low concentrations such that it is possible for individual polynucleotides to be located within individual polymer matrices of the array of polymer matrices. The polymer matrix may be exposed to enzymes, nucleotides, salts, or other components sufficient to promote replication of the target polynucleotide.

In particular embodiments, an enzyme, such as a polymerase, is present in, attached to, or in close proximity to the polymer matrix. A variety of nucleic acid polymerases can be used in the methods described herein. In exemplary embodiments, the polymerase may include an enzyme, fragment or subunit thereof that may catalyze replication of a polynucleotide. In another embodiment, the polymerase may be a naturally occurring polymerase, a recombinant polymerase, a mutant polymerase, a variant polymerase, a fused or otherwise engineered polymerase, a chemically modified polymerase, a synthetic molecule, or an analog, derivative, or fragment thereof.

In some embodiments, methods for partitioning a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprise a nucleic acid amplification reaction. In some embodiments, any type of nucleic acid amplification reaction may be performed, including Polymerase Chain Reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis), ligase Chain Reaction (LCR) (Barany 1991Proceedings National Academy of Science USA 88:189-193;Barnes 1994Proceedings National Academy of Science USA91:2216-2220), helicase-dependent amplification (HDA), or isothermal self-sustaining sequence reaction (Kwoh 1989Proceedings National Academy of Science USA86:1173-1177; WO 1988/10315; and U.S. Pat. Nos. 5,409,818,5,399,491 and 5,194,370).

In some embodiments, the amplification reaction comprises Recombinase Polymerase Amplification (RPA). (see, e.g., U.S. Pat. No. 5,223,414 to Zarling, U.S. Pat. Nos. 5,273,881 and 5,670,316 to Sena, and U.S. Pat. Nos. 7,270,981, 7,399,590, 7,435,561, 7,666,598, 7,763,427, 8,017,339, 8,030,000, 8,062,850, and 8071308).

In some embodiments, the method for partitioning a single target polynucleotide into a reaction chamber and amplifying a single target polynucleotide comprises isothermal amplification conditions. In some embodiments, the nucleic acid amplification reaction may be performed under isothermal conditions. In some embodiments, isothermal amplification conditions include nucleic acid amplification reactions that undergo such temperature changes: the temperature change is limited to a limited range during at least a portion of the amplification, including, for example, a temperature change within about 20 ℃, or about 10 ℃, or about 5 ℃, or about 1-5 ℃, or about 0.1-1 ℃, or less than about 0.1 ℃. In some embodiments, the nucleic acid amplification reaction may be performed under isothermal or thermocycling conditions.

In some embodiments, nucleic acids that have been amplified according to the present teachings can be used in any nucleic acid sequencing workflow, including sequencing by ligation and detection of oligonucleotide probes (e.g., SOLiD from Life Technologies ^TM WO 2006/084131), probe-anchored ligation sequencing (e.g. Complete Genomics ^TM Or a Polonator ^TM ) Sequencing by synthesis (e.g., genetic Analyzer and HiSeq from Illumina ^TM ) Pyrophosphate sequencing (e.g., genome Sequencer FLX from 454Life Sciences), ion-sensitive sequencing (e.g., personal Genome Machine (PGM) from Ion Torrent Systems, inc ^TM ) And Ion Proton ^TM Sequencer) and single molecule sequencing platform (e.g., from Helicos) ^TM HeliScope of (A) ^TM )。

In some embodiments, nucleic acids that have been amplified according to the present teachings can be sequenced by any sequencing method, including sequencing by synthesis, ion-based sequencing including detection of sequencing byproducts using field effect transistors (e.g., FETs and ISFETs), chemical degradation sequencing, ligation-based sequencing, sequencing by hybridization, pyrophosphate detection sequencing, capillary electrophoresis, gel electrophoresis, next generation, massively parallel sequencing platforms, sequencing platforms that detect hydrogen ions or other sequencing byproducts, and single molecule sequencing platforms. In some embodiments, the sequencing reaction may be performed using at least one sequencing primer that hybridizes to any portion of the polynucleotide construct, including the nucleic acid linker or the target polynucleotide.

In some embodiments, a method of detecting one or more byproducts of nucleotide incorporation may be used to sequence a nucleic acid amplified according to the present teachings. Polymerase extension detection by detecting physicochemical byproducts of the extension reaction may include pyrophosphate, hydrogen ions, charge transfer, heat, and the like, as disclosed, for example, in U.S. patent No. 7,948,015 to Rothberg et al and U.S. patent publication No. 2009/0026082 to Rothberg et al (incorporated herein by reference in its entirety). Other examples of methods for detecting polymerase-based extension can be found, for example, in poulmand et al, proc.Natl. Acad.Sci.,103:6466-6470 (2006); purushaman et al IEEE ISCAS, IV-169-172; anderson et al, sensors and Actuators B chem.,129:79-86 (2008); sakata et al, angew.chem.118:2283-2286 (2006); esfandyapour et al, U.S. patent publication No. 2008/01666727; and Sakurai et al, anal chem.64:1996-1997 (1992).

Reactions involving the generation and detection of ions are widely performed. Monitoring the progress of such reactions using direct ion detection methods can simplify many current bioassays. For example, template-dependent nucleic acid synthesis mediated by a polymerase can be monitored by detecting hydrogen ions generated as a natural byproduct of nucleotide incorporation catalyzed by the polymerase. Ion sensitive sequencing (also known as "pH-based" or "ion-based" nucleic acid sequencing) utilizes direct detection of ionic byproducts such as hydrogen ions (produced as a byproduct of nucleotide incorporation). In one exemplary system for ion-based sequencing, the nucleic acid to be sequenced can be captured in a microwell and the nucleotides flowed through the well one at a time under nucleotide incorporation conditions. The polymerase incorporates the appropriate nucleotides into the growing strand and the released hydrogen ions can change the pH of the solution, which can be detected by an ion sensor coupled to the well. This technique does not require labeling of nucleotides or expensive optical components and allows for much faster completion of sequencing runs. Examples of such Ion-based nucleic acid sequencing methods and platforms include Ion Torrent PGM ^TM Or Proton ^TM Sequencer (Ion Torrent) ^TM Systems,Life Technologies Corporation)。

In some embodiments, target polynucleotides produced using the methods, systems, and kits of the present teachings can be used as substrates for biological or chemical reactions that are detected and/or monitored by sensors, including Field Effect Transistors (FETs). In various embodiments the FET is a chemFET or ISFET. "chemFET" or chemical field effect transistor is a type of field effect transistor used as a chemical sensor. It is a structural analogue of a MOSFET transistor in which the charge on the gate electrode is applied by a chemical process. "ISFET" or ion sensitive field effect transistor is used to measure the concentration of ions in a solution; when the ion concentration (e.g., h+) changes, the current through the transistor will change accordingly. A detailed theory of operation of ISFETs is found in "Thirty years of ISFETOLOGY: what happened in the past 30years and what may happen in the next 30years," P.Bergveld, sens.Actuators,88 (2003), pp.1-20.

In some embodiments, the FET may be an array of FETs. As used herein, an "array" is a planar arrangement of elements such as sensors or wells. The array may be one-dimensional or two-dimensional. A one-dimensional array may be an array having one column (or row) of elements in a first dimension and a plurality of columns (or rows) in a second dimension. The number of columns (or rows) in the first and second dimensions may or may not be the same. The FET or array may comprise 102, 103, 104, 105, 106, 107 or more FETs.

In some embodiments, one or more microfluidic structures may be soldered over the FET sensor array to provide containment and/or enclosure of biological or chemical reactions. For example, in one instrument, the microfluidic structure may be configured as one or more wells (or microwells or reaction chambers or reaction wells, these terms being used interchangeably herein) disposed over one or more sensors of the array, such that the one or more sensors on which a given well is disposed detect and measure the presence, level, and/or concentration of an analyte in the given well. In some embodiments, the correspondence of FET sensors and reaction wells may be 1:1.

The microwells or reaction chambers are typically holes or pores having well-defined shapes and volumes that can be machined into the substrate and welded using conventional micro-welding techniques, such as disclosed in the following references: doering and Nishi, editors, handbook of Semiconductor Manufacturing Technology, second Edition (CRC Press, 2007); saliterman, fundamentals of BioMEMS and Medical Microdevices (SPIE Publications, 2006); elwensporek et al, silicon Micromachining (Cambridge University Press, 2004); and the like. Examples of structures (e.g., spacing, shape, and volume) of microwells or reaction chambers are disclosed in U.S. patent publication 2009/012589 to Rothberg et al; british patent application GB24611127 to Rothberg et al.

In some embodiments, the biological or chemical reaction may be performed in a solution or reaction chamber in contact with, operatively coupled to, or capacitively coupled to a FET, such as a chemFET or ISFET. The FET (or chemFET or ISFET) and/or the reaction chamber may be an array of FETs or reaction chambers, respectively.

In some embodiments, the biological or chemical reaction may be performed in a two-dimensional array of reaction chambers, where each reaction chamber may be coupled to a FET and each reaction chamber is no more than 10 μm in capacity ³ (i.e., 1 pL). In some embodiments, each reaction chamber is no more than 0.34pL, 0.096pL, or 0.012pL in capacity. The reaction chamber may optionally be no more than 2, 5, 10, 15, 22, 32, 42, 52, 62, 72, 82, 92, or 102 square microns in cross-sectional area at the top. Preferably, the array has at least 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ Or more reaction chambers. In some embodiments, at least one of the reaction chambers is operatively coupled to at least one of the FETs.

The FET arrays for use in accordance with various embodiments of the present disclosure may be soldered according to conventional CMOS soldering techniques as well as modified CMOS soldering techniques and semiconductor soldering techniques other than those conventionally used in CMOS soldering. In addition, various lithographic techniques may be used as part of the array soldering process.

Exemplary FET arrays and microwells and fluids suitable for use in the disclosed methods and methods of making the same are disclosed, for example, in U.S. patent publication No. 20100301398; U.S. patent publication No. 20100300895; U.S. patent publication No. 20100300559; U.S. patent publication No. 20100197507, U.S. patent publication No. 20100137143; U.S. patent publication No. 20090127589; and U.S. patent publication No. 200990026082, the above references are incorporated by reference in their entirety.

In one aspect, the disclosed methods, compositions, systems, devices, and kits can be used to perform label-free nucleic acid sequencing, particularly ion-based nucleic acid sequencing. The concept of label-free detection of nucleotide incorporation has been described in the literature, including the following (which is incorporated herein by reference): U.S. patent publication 2009/0026082 to Rothberg et al; anderson et al, sensors and Actuators B chem.,129:79-86 (2008); and Pourmand et al, proc.Natl.Acad.Sci.,103:6466-6470 (2006). Briefly, in nucleic acid sequencing applications, nucleotide incorporation is determined by measuring natural byproducts of polymerase-catalyzed extension reactions including hydrogen ions, polyphosphates, PPi, and Pi (e.g., in the presence of pyrophosphatase). Examples of such Ion-based nucleic acid sequencing methods and platforms include Ion Torrent PGM ^TM Or Proton ^TM Sequencer (Ion Torrent) ^TM Systems,Life Technologies Corporation)。

In some embodiments, the present disclosure generally relates to methods for sequencing nucleic acids that have been amplified by the teachings provided herein. In one exemplary embodiment, the present disclosure relates generally to a method for obtaining sequence information from a polynucleotide, comprising: (a) amplifying the nucleic acid; and (b) performing template-dependent nucleic acid synthesis using at least one of the amplified nucleic acids generated during step (a) as a template. Amplification may optionally be performed according to any of the amplification methods described herein.

In some embodiments, the template-dependent synthesis includes incorporating one or more nucleotides into the newly synthesized nucleic acid strand in a template-dependent manner.

Optionally, the method may further comprise generating one or more ionic byproducts of such nucleotide incorporation.

In some embodiments, the method may further comprise detecting incorporation of one or more nucleotides into the sequencing primer. Optionally, detecting may include detecting release of hydrogen ions.

In another embodiment, the present disclosure generally relates to a method for sequencing a nucleic acid, comprising: (a) amplifying the nucleic acid according to the methods disclosed herein; (b) The amplified nucleic acids are placed in a plurality of reaction chambers, wherein one or more of the reaction chambers are in contact with a Field Effect Transistor (FET). Optionally, the method further comprises contacting the amplified nucleic acid disposed in one of the reaction chambers with a polymerase, thereby synthesizing a new nucleic acid strand by sequentially incorporating one or more nucleotides into the nucleic acid molecule. Optionally, the method further comprises generating one or more hydrogen ions as a byproduct of the incorporation of such nucleotides. Optionally, the method further comprises detecting incorporation of the one or more nucleotides by detecting production of the one or more hydrogen ions using the FET.

In some embodiments, detecting includes detecting a change in voltage and/or current at least one FET within the array in response to the generation of one or more hydrogen ions.

In some embodiments, the FET may be selected from: ion sensitive FETs (isfets) and chemosensitive FETs (chemfets).

One exemplary system that includes sequencing of ionic byproducts incorporated by detection of nucleotides is Ion Torrent PGM ^TM Or Proton ^TM A sequencer (Life Technologies) is an ion-based sequencing system that sequences nucleic acid templates by detecting hydrogen ions generated as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as nucleotide incorporation byproducts that are present during polymerase-mediated template-dependent nucleic acid synthesis. Ion Torrent PGM ^TM Or Proton ^TM The sequencer detects nucleotide incorporation by detecting hydrogen ion byproducts of the nucleotide incorporation. Ion Torrent PGM ^TM Or Proton ^TM The sequencer may comprise a plurality of nucleic acid templates to be sequenced, wherein each template is placed within a respective sequencing reaction well in the array. The wells of the array may each be coupled to at least one ion sensor that can detect H generated as a byproduct of nucleotide incorporation ⁺ Release or of ions The pH of the solution changes. The ion sensor includes a sensor coupled to an inductively-sensitive H ⁺ A Field Effect Transistor (FET) of the ion sensitive detection layer that detects the presence of ions or changes in the pH of the solution. The ion sensor may provide an output signal indicative of nucleotide incorporation, which may be expressed as a voltage change of a magnitude that is consistent with H in the respective well or reaction chamber ⁺ Ion concentration is related. The different nucleotide types may flow continuously into the reaction chamber and may be incorporated into the extension primer (or polymerization site) by the polymerase in an order determined by the sequence of the template. Each nucleotide incorporation may be accompanied by H in the reaction well ⁺ The release of ions and the concurrent change of local pH. H ⁺ The release of ions may be recorded by the FET of the sensor, which generates a signal indicative of nucleotide incorporation occurring. Nucleotides that are not incorporated in a particular nucleotide stream may not generate a signal. The amplitude of the signal from the FET can also be related to the number of specific types of nucleotides incorporated into the extended nucleic acid molecule, allowing for resolution of the homopolymer region. Thus, during operation of the sequencer, multiple nucleotide flows into the reaction chamber and incorporation monitoring between a large number of wells or reaction chambers may allow the device to simultaneously parse the sequences of many nucleic acid templates. Regarding Ion Torrent PGM ^TM Or Proton ^TM Additional details of sequencer composition, design, and operation can be found, for example, in U.S. patent application Ser. No. 12/002781, now disclosed as U.S. patent publication No. 2009/0026082; U.S. patent application Ser. No. 12/474897, now published as U.S. patent publication No. 2010/0137443; and U.S. patent application Ser. No. 12/492844, now disclosed as U.S. patent publication No. 2010/0282617, the above references are incorporated by reference in their entirety.

FIG. 28 shows a block diagram of components of a system for nucleic acid sequencing according to an exemplary embodiment. The components include flow cell 101, reference electrode 108, a plurality of reagents 114 for sequencing, valve body 116, wash solution 110, valve 112, fluid controller 118, lines 120/122/126, channels 104/109/111, waste container 106, array controller 124, and user interface 128 on integrated circuit device 100. The integrated circuit device 100 includes a microwell array 107 overlying a sensor array that includes chemical sensors as described herein. The flow cell 101 comprises an inlet 102, an outlet 103 and a flow chamber 105 defining a reagent flow path over a microwell array 107.

Reference electrode 108 can be of any suitable type or shape, including a coaxial cylinder with a flow channel or a wire inserted into the lumen of channel 111. Reagent 114 may be driven through flow paths, valves, and flow cell 101 by a pump, pneumatic pressure, or other suitable method, and may be discharged into waste container 106 after exiting outlet 103 of flow cell 101. The fluid controller 118 may control the actuation force for the reagent 114, the operation of the valve 112 and the valve body 116 using appropriate software.

Microwell array 107 includes an array of reaction zones as described herein, also referred to herein as microwells, operably associated with corresponding chemical sensors in a sensor array. For example, each reaction zone may be coupled to a chemical sensor suitable for detecting an analyte of interest or a reaction property within the reaction zone. The micro-hole array 107 may be integrated into the integrated circuit device 100 such that the micro-hole array 107 and the sensor array are part of a single device or chip.

Flow cell 101 can have a variety of structures for controlling the path and flow rate of reagents 114 over microwell array 107. The array controller 124 provides bias voltages and timing and control signals to the integrated circuit device 100 for reading the chemical sensors of the sensor array. Array controller 124 also provides a reference bias voltage to reference electrode 108 to bias reagent 114 flowing through microwell array 107.

During an experiment, array controller 124 collects and processes output signals from chemical sensors of the sensor array through output ports on integrated circuit device 100 via bus 127. The array controller 124 may be a computer or other computing tool. The array controller 124 may include memory and application software for storing data, a processor for accessing data and executing applications, and components that facilitate communication with the various components of the system in fig. 28.

The value of the output signal of the chemical sensor is indicative of a physical and/or chemical parameter of one or more reactions occurring in a corresponding reaction zone in the microwell array 107. For example, in an exemplary embodiment, the values of the output signal may be processed using techniques disclosed in the following references: U.S. patent application Ser. No. 13/339,846 to Rearick et al, 12.29.2011, U.S. provisional patent application Ser. No. 61/428,743 to 30.2010, U.S. provisional patent application Ser. No. 61/429,328 to 1.3.2011, and U.S. patent application Ser. No. 13/339,753 to Hubbell, 12.29.2011, U.S. provisional patent application Ser. No. 61/428,097 to 29.2010.

The user interface 128 may display information regarding the flow cell 101 and output signals received from chemical sensors in the sensor array on the integrated circuit device 100. The user interface 128 may also display device settings and controls and allow a user to input or set device settings and controls.

In an exemplary embodiment, the fluid controller 118 may control the delivery of the individual reagents 114 to the flow cell 101 and integrated circuit device 100 in a predetermined sequence, for a predetermined duration, at a predetermined flow rate during the experiment. The array controller 124 may then collect and analyze output signals of the chemical sensors indicative of chemical reactions occurring in response to the delivery of the reagent 114.

During the experiment, the system may also monitor and control the temperature of the integrated circuit device 100 so that reactions occur and measurements are made at known, predetermined temperatures.

The system can be configured such that a single fluid or reagent contacts the reference electrode 108 throughout a multi-step reaction during operation. Valve 112 may be closed to prevent any wash solution 110 from flowing into channel 109 as reagent 114 flows. Although the flow of the wash solution may be prevented, there may still be continuous fluid and electrical communication between the reference electrode 108, the channel 109, and the microwell array 107. The distance between reference electrode 108 and the junction between channels 109 and 111 may be selected so that no or very little of the reagent flowing in channel 109 and possibly diffusing into channel 111 reaches reference electrode 108. In an exemplary embodiment, the wash solution 110 can be selected to be in continuous contact with the reference electrode 108, which can be particularly useful for multi-step reactions that use frequent wash steps.

Fig. 29 shows a cross-sectional view and an expanded view of a portion of the integrated circuit device 100 and the flow cell 101. During operation, the flow cell 101 flow chamber 105 encloses a reagent stream 208 of delivered reagent flowing through the open end of the reaction zone in the microwell array 107. The volume, shape, aspect ratio (e.g., bottom width to hole depth ratio) and other dimensional characteristics of the reaction zone may be selected based on the nature of the reaction that occurs and the labeling technique (if any) or reagents, byproducts used.

The chemical sensors of the sensor array 205 sense (and generate output signals) chemical reactions within the associated reaction zones in the microwell array 107 to detect an analysis of interest or a property of the reaction. The chemical sensors of sensor array 205 may be, for example, chemosensitive field effect transistors (chemfets), such as Ion Sensitive Field Effect Transistors (ISFETs). Examples of chemical sensors and array structures that may be used in embodiments are described in U.S. patent application publication nos. 2010/0300559, 2010/0197507, 2010/0301398, 2010/0300895, 2010/0137443 and 2009/0026082, and U.S. patent No. 7,575,865, each of which is incorporated herein by reference.

FIG. 30 shows a cross-sectional view of two representative chemical sensors and their corresponding reaction zones, according to an example embodiment. In fig. 30, two

chemical sensors

350, 351 are shown, representing a small portion of a sensor array that may include millions of chemical sensors. In some embodiments, the sensor array may include at least 1 million chemical sensors and optionally at least 1 million corresponding reaction zones, at least 5 million chemical sensors and optionally at least 5 million corresponding reaction zones or at least 10 million chemical sensors and optionally at least 10 million corresponding reaction zones.

Chemical sensor 350 is coupled to a corresponding reaction zone 301 and chemical sensor 351 is coupled to a corresponding reaction zone 302. Chemical sensor 350 represents a chemical sensor in a sensor array. In the example shown, chemical sensor 350 is an ion sensitive field effect transistor. Chemical sensor 350 includes a floating gate structure 318, which floating gate structure 318 has a floating gate conductor (referred to herein as a sensor plate 320) separated from reaction region 301 by a sensing material 316. As shown in fig. 30, the sensor plate 320 is the uppermost patterned layer of conductive material in the floating gate structure 318 under the reaction region 301.

In the example shown, floating gate structure 318 includes multiple patterned layers of conductive material in layers of dielectric material 319. As described in more detail below, the upper surface of the sensing material 316 serves as the sensing surface 317 of the chemical sensor 350.

In the embodiment shown, the sensing material 316 is an ion sensitive material such that the presence of ions or other charge species in solution in the reaction zone 301 alters the surface potential of the sensing surface 317. The change in surface potential is caused by the protonation or deprotonation of the surface charge groups at the sensing surface due to ions present in the solution. The sensing material 316 may be embedded using a variety of techniques or formed naturally during one or more manufacturing processes used to form the chemical sensor 350. In some embodiments, the sensing material 316 is a metal oxide, such as an oxide of silicon, tantalum, aluminum, lanthanum, titanium, zirconium, hafnium, tungsten, palladium, iridium, or the like.

In some embodiments, the sensing material 316 is an oxide of an upper layer of conductive material of the sensor plate 320. For example, the upper layer of the sensor plate 320 may be titanium nitride and the sensing material 316 may include titanium oxide or titanium oxynitride. More generally, the sensing material 316 may include one or more of a variety of different materials to facilitate sensitivity to particular ions. For example, silicon nitride or silicon oxynitride and metal oxides such as silicon oxide, aluminum oxide or tantalum oxide generally provide sensitivity to hydrogen ions, whereas sensing materials comprising polyvinylchloride with valinomycin provide sensitivity to potassium ions. Depending on the instrument, materials that are sensitive to other ions (e.g., sodium, silver, iron, bromine, iodine, calcium, and nitrate) may also be used.

The chemical sensor 350 also includes a source region 321 and a drain region 322 within a semiconductor substrate 354. The source region 321 and the drain region 322 comprise a doped semiconductor material having a conductivity type that is different from the conductivity type of the substrate 354. For example, the source region 321 and the drain region 322 may comprise a doped P-type semiconductor material and the substrate may comprise a doped N-type semiconductor material.

The trench region 323 separates the source region 321 and the drain region 322. Floating gate structure 318 overlies trench region 323 and is separated from substrate 354 by gate dielectric 352. Gate dielectric 352 may be, for example, silicon dioxide. Alternatively, other dielectrics may be used for gate dielectric 352.

As shown in fig. 30, the reaction zone 301 extends through the fill material 310 on the dielectric material 319. The fill material 310 may, for example, comprise one or more layers of dielectric material such as silicon dioxide or silicon nitride.

The dimensions (e.g., width and depth) of the

reaction zones

301, 302 and their pitch (center distance between adjacent reaction zones) may vary from instrument to instrument. In some embodiments, the reaction zone may have a characteristic diameter of no more than 5 microns, such as no more than 3.5 microns, no more than 2.0 microns, no more than 1.6 microns, no more than 1.0 microns, no more than 0.8 microns, no more than 0.6 microns, no more than 0.4 microns, no more than 0.2 microns, or no more than 0.1 microns, the diameter being defined by 4 times the plan view cross-sectional area (a) divided by the square root of Pi (e.g., sqrt (4*A/Pi)).

In some embodiments, the pitch between adjacent reaction zones is no more than 10 microns, no more than 5 microns, no more than 2 microns, no more than 1 micron, or no more than 0.5 microns.

In the embodiment shown, the

reaction zones

301, 302 are separated by a distance equal to their width. Alternatively, the separation distance between adjacent reaction zones may be less than its width. For example, the separation distance may be the minimum feature size for the process used to form the reaction zones 301, 302 (e.g., a lithographic process). In such cases, the separation may be directly significantly less than the width of the individual reaction zones.

The sensor plate 320, the sensing material 316, and the reaction zone 301 may, for example, have a circular cross-section. Alternatively, these may be non-circular. For example, the cross-section may be square, rectangular, hexagonal, or irregularly shaped.

Depending on the device and array structure in which the chemical sensors described herein are implemented, the device in fig. 30 may also include additional elements such as array lines (e.g., word lines, bit lines, etc.) for accessing the chemical sensors, additional doped regions in the substrate 354, and other circuitry (e.g., access circuitry, bias circuitry, etc.) for operating the chemical sensors. In some embodiments, devices may be produced, for example, using techniques described in U.S. patent application publication nos. 2010/0300559, 2010/0197507, 2010/0301398, 2010/0300895, 2010/013743, and 2009/0026082, and U.S. patent No. 7,575,865 (each of which is incorporated herein by reference).

In operation, reactants, wash solutions, and other reagents can move into and out of the reaction zone 301 via the diffusion mechanism 340. The chemical sensor 350 senses (and generates an associated output signal) the amount of charge 324 present on the sensing material 316 opposite the sensor plate 320. The change in charge 324 causes a change in voltage on floating gate structure 318, which in turn causes a change in the threshold voltage of the transistor. The change in threshold voltage may be measured by measuring the current in the tub region 323 between the source region 321 and the drain region 322. Thus, chemical sensor 350 may be used directly to provide a current-based output signal on an array line connected to source region 321 or drain region 322, or indirectly with additional circuitry to provide a voltage-based output signal.

In embodiments, the reaction performed in reaction zone 301 may be an analytical reaction to identify or determine a characteristic or property of an analyte of interest. Such a reaction may directly or indirectly produce byproducts that affect the amount of charge near the sensor plate 320. If such byproducts are generated in small amounts or decay rapidly or react with other components, multiple copies of the same analyte may be analyzed simultaneously in reaction zone 301 to increase the output signal generated. In embodiments, multiple copies of the analyte may be attached to the solid support 312 either before or after placement in the reaction zone 301. The solid support 312 may be microparticles, nanoparticles, beads, solid or porous including gels, and the like. For simplicity and ease of description, the solid support 312 is also referred to herein as a particle. For nucleic acid analytes, multiple linked copies can be made by Rolling Circle Amplification (RCA), exponential RCA, recombinase Polymerase Amplification (RPA), polymerase chain reaction amplification (PCR), emulsion PCR amplification, or similar techniques to produce amplicons without the need for a solid support.

In various exemplary embodiments, the methods, systems, and computer-readable media described herein may be advantageously used to process and/or analyze data and signals obtained from electronic or charge-based nucleic acid sequencing. In electron-or charge-based sequencing (e.g., pH-based sequencing), nucleotide incorporation events can be determined by detecting ions (e.g., hydrogen ions) generated as a natural byproduct of a polymerase-catalyzed nucleotide extension reaction. This can be used to sequence a sample or template nucleic acid, which can be, for example, a fragment of a nucleic acid sequence of interest, and which can be directly or indirectly attached as a clonal population to a solid support such as a particle, microparticle, bead, or the like. The sample or template nucleic acid may be operably associated to the primer and polymer and may undergo repeated cycles or "flows" of nucleotide addition and washing (which may be referred to herein as "nucleotide flows" which may result in nucleotide incorporation). The primer may be annealed to the sample or template so that the 3' end of the primer may be extended by the polymerase whenever a nucleotide complementary to the next base in the template is added. Subsequently, based on the sequences of the known nucleotide streams and based on the measured output signals of the chemical sensors indicative of the ion concentration during each nucleotide stream, information of the type, sequence and number of nucleotides associated with the sample nucleic acid present in the reaction zone coupled to the chemical sensor can be determined.

In typical ion-based nucleic acid sequencing embodiments, nucleotide incorporation can be detected by detecting the presence and/or concentration of hydrogen ions generated by a polymerase-catalyzed extension reaction. In one embodiment, templates optionally pre-bound to sequencing primers and/or polymerase may be loaded into a reaction chamber (e.g., microwells as disclosed in Rothberg et al cited herein), followed by repeated cycles of nucleotide addition and washing. In some embodiments, such templates may be attached as clonal populations to a solid support such as a particle, bead, or the like, and the clonal populations loaded into a reaction chamber.

In another embodiment, templates optionally bound to a polymerase are assigned to, placed in, or located at different sites of the array. The sites of the array comprise primers and the method may comprise hybridizing different templates to the primers in different sites.

In each addition step of the cycle, the polymerase can extend the primer by incorporating the added nucleotide only if the next base in the template is the complement of the added nucleotide. If there is one complementary base, there is one incorporation, if there are two, if there are three, etc. For each such incorporation, there are released hydrogen ions, and the population of templates that collectively release hydrogen ions change the local pH of the reaction chamber. The generation of hydrogen ions is monotonically related to the number of consecutive complementary bases in the template (and to the total number of template molecules that the primer and polymer participate in the extension reaction). Thus, when there are a number of consecutive identical complementary bases (i.e., homomeric regions) in the template, the amount of hydrogen ions generated and thus the magnitude of the local pH change can be proportional to the number of consecutive identical complementary bases. If the next base in the template is not complementary to the added nucleotide, then incorporation does not occur and no hydrogen ions are released. In some embodiments, after each step of adding a nucleotide, an additional step may be performed in which a buffer-free wash solution at a predetermined pH is used to remove the nucleotides of the previous step to prevent misincorporation in later cycles. In some embodiments, after each step of adding a nucleotide, an additional step may be performed in which the reaction chamber is treated with a nucleotide disrupting agent, such as apyrase, to remove any remaining nucleotides remaining in the chamber, which may result in pseudo-extension in subsequent cycles.

In one exemplary embodiment, different kinds of nucleotides are added to the reaction chamber consecutively, so that each reaction can be exposed to different nucleotides one at a time. For example, nucleotides may be added in the following order: dATP, dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, etc.; each exposure is followed by a washing step. The loop may be repeated 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 750 times or more depending on the length of the desired sequence information.

In some embodiments, may be in accordance with PGM ^TM Or Proton ^TM The user manual provided by the sequencer was used to sequence. Example 3 provides a method for using Ion Torrent PGM ^TM Sequencer (Ion Torrent) ^TM Systems, life Technologies, CA).

In some embodiments, the disclosure generally relates to methods for sequencing a population of template polynucleotides, comprising: (a) Generating a plurality of amplicons by clonally amplifying a plurality of template polynucleotides on a plurality of surfaces, wherein the amplifying is performed within a single continuous phase of a reaction mixture and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the amplicons generated are substantially monoclonal in nature. In some embodiments of the present invention, in some embodiments, generating a sufficient number of basic species in a single amplification reaction the amplicon was then cloned into Ion Torrent PGM ^TM 314. 316 or 318 sequencer produced at least 100MB, 200MB, 300MB, 400MB, 500MB, 750MB, 1GB or 2GB AQ20 sequencing reads. As used herein, the term "AQ20" and variants thereof refers to PGM at Ion Torrent ^TM Specific methods of measuring sequencing accuracy in sequencers. Accuracy can be measured in terms of a Phred-sample Q score that measures accuracy on a logarithmic scale: q10=90%, q20=99%, q30=99.9%, q40=99.99% and q50=99.999%. For example, in a particular sequencing reaction, the accuracy metric may be calculated by a predictive algorithm or by actual alignment with a known reference genome. The predicted quality score ("Q score") may be derived from the algorithm: which takes into account the inherent nature of the input signal and whether a given single base contained in a sequencing "read" will pairA fairly accurate evaluation was performed. In some embodiments, such predicted mass fractions may be used to filter and remove low quality readings prior to downstream alignment. In some embodiments, accuracy may be reported in terms of a Phred-like Q score that measures accuracy on a logarithmic scale: q10=90%, q17=98%, q20=99%, q30=99.9%, q40=99.99% and q50=99.999%. In some embodiments, the data obtained from a given polymerase reaction may be filtered to measure only polymerase readings measuring "N" nucleotides or more and having a Q score above a certain threshold, e.g., Q10, Q17, Q100 (referred to herein as the "NQ17" score). For example, a 100Q20 score may indicate the number of reads obtained from a given reaction that are at least 100 nucleotides in length and have a Q20 (99%) or higher Q score. Similarly, a 200Q20 score may indicate the number of reads that are at least 200 nucleotides in length and have a Q score of Q20 (99%) or higher.

In some embodiments, accuracy, which is referred to herein as "raw" accuracy, may also be calculated based on the appropriate alignment using the reference genomic sequence. This is a single pass accuracy (single pass accuracy) that includes a "true" measurement of per base error associated with a single reading, as opposed to a common accuracy that measures the error rate from a common sequence, which is the result of multiple readings. The raw accuracy measurement may be reported in terms of an "AQ" ("quality of comparison" shorthand) score. In some embodiments, the data obtained from a given polymerase reaction may be filtered to measure only polymerase readings measuring "N" nucleotides or more and having AQ scores exceeding certain thresholds, e.g., AQ10, AQ17, AQ100 (referred to herein as "NAQ17" scores). For example, a 100AQ20 score may indicate the number of reads obtained from a given polymerase reaction that are at least 100 nucleotides in length and have an AQ20 (99%) or higher. Similarly, a 200AQ20 score may indicate the number of reads that are at least 200 nucleotides in length and have an AQ20 (99%) or higher.

In some embodiments, the present teachings provide a system for nucleic acid amplification comprising any combination of the following: beads to which a plurality of first primers are attached, a second primer, a third primer, a polynucleotide, a recombinase-loaded protein, a single-stranded binding protein (SSB), a polymerase, nucleotides, ATP, phosphocreatine, creatine kinase, a hybridization solution, and/or a wash solution. The system may comprise all or some of these components. In some embodiments, the system for nucleic acid amplification may further comprise any combination of buffers and/or cations (e.g., divalent cations).

In some embodiments, the present teachings provide kits for nucleic acid amplification. In some embodiments, the kit comprises any reagents useful for nucleic acid amplification. In some embodiments, the kit may comprise any combination of the following: beads to which a plurality of first primers are attached, a second primer, a third primer, a polynucleotide, a recombinase-loaded protein, a single-stranded binding protein (SSB), a polymerase, nucleotides, ATP, phosphocreatine, creatine kinase, hybridization solution, wash solution, buffer, and/or cation (e.g., divalent cation). The kit may contain all or some of these components.

In some embodiments, the present disclosure generally relates to methods, compositions, systems for amplifying different nucleic acid templates in parallel in multiple partitioned reaction volumes (as opposed to amplification within a single continuous liquid phase). For example, the nucleic acid templates may be distributed or placed into an array of reaction chambers or an array of reaction volumes such that at least two such chambers or volumes in the array each receive a single nucleic acid template. In some embodiments, a plurality of separate reaction volumes are formed. The reaction chamber (or reaction volume) may optionally be closed prior to amplification. In another embodiment, the reaction mixture may be divided or separated into a plurality of microreactors dispersed within a continuous phase of the emulsion. The partitioned or separated reaction volumes optionally do not mix or communicate with each other or are not capable of mixing or communicating with each other. In some embodiments, at least some of the reaction chambers (or reaction volumes) comprise a recombinase and optionally a polymerase. The polymerase may be a strand displacement polymerase.

In some embodiments, the present disclosure relates generally to compositions, systems, methods, devices, and kits comprising emulsions for nucleic acid synthesis and/or amplification. As used herein, the term "emulsion" includes any composition comprising a mixture of a first liquid and a second liquid, wherein the first and second liquids are substantially immiscible with each other. Typically, one of the liquids is hydrophilic while the other liquid is hydrophobic. Typically, emulsions comprise a dispersed phase and a continuous phase. For example, the first liquid may form a dispersed phase dispersed in a second liquid that forms a continuous phase. The dispersed phase optionally consists essentially of the first liquid. The continuous phase optionally consists essentially of the second liquid. In various embodiments, the same two liquids may form different types of emulsions. For example, a mixture comprising oil and water may first form an oil-in-water emulsion, where the oil is the dispersed phase and the water is the dispersing medium. Second, they can form water-in-oil emulsions, where water is the dispersed phase and oil is the external phase. Multiple emulsions are also possible, including "water-in-oil-in-water" emulsions and "oil-in-water-in-oil" emulsions. In some embodiments, the dispersed phase comprises one or more microreactors in which the nucleic acid templates can be individually amplified. One or more microreactors may form a partitioned reaction volume in which separate amplification reactions may occur. One example of a suitable vehicle for nucleic acid amplification includes a water-in-oil emulsion, wherein the water-based phase comprises several aqueous microreactors dispersed within the oil phase of the emulsion. In some embodiments, the emulsion may further comprise an emulsifier or surfactant. Emulsifiers or surfactants may be used to stabilize the emulsion under nucleic acid synthesis conditions.

In some embodiments, the present disclosure generally relates to compositions comprising an emulsion comprising a reaction mixture. The emulsion may comprise an aqueous phase. The aqueous phase may be dispersed in the continuous phase of the emulsion. The aqueous phase may comprise one or more microreactors. In some embodiments, the reaction mixture is contained in a plurality of liquid-phase microreactors within a phase of the emulsion. Optionally, the reaction mixture comprises a recombinase. Optionally, the reaction mixture comprises a plurality of different polynucleotides. Optionally, the reaction mixture comprises a plurality of supports. Optionally, the reaction mixture comprises any combination of a recombinase, a plurality of different polynucleotides, and/or a plurality of supports. Optionally, at least one support may be attached to a substantially monoclonal population of nucleic acids.

In some embodiments, the present disclosure generally relates to a composition comprising a reaction mixture comprising (i) a plurality of supports, (ii) a plurality of different polynucleotides, and (iii) a recombinase enzyme, the reaction mixture being contained in a plurality of liquid-phase microreactors in an emulsion.

In some embodiments, the present disclosure generally relates to a composition comprising a reaction mixture comprising (i) a recombinase enzyme and (ii) a plurality of supports, at least one support being connectable to a substantially monoclonal nucleic acid population, wherein the reaction mixture is comprised in a plurality of liquid-phase microreactors in an emulsion.

Optionally, the emulsion comprises a hydrophilic phase.

Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. For example, the emulsion may comprise a water-in-oil emulsion.

In some embodiments, the hydrophilic phase comprises a plurality of microreactors.

Optionally, the reaction mixture is contained in a single reaction vessel.

Optionally, the sequences of a plurality of different polynucleotides may be the same or different.

Optionally, at least one of the plurality of supports is linked to a plurality of first primers (e.g., forward amplification primers).

Optionally, the reaction mixture further comprises a plurality of second primers (e.g., reverse amplification primers).

In some embodiments, at least one of the plurality of supports further comprises a plurality of second primers.

In some embodiments, at least one of the plurality of supports comprises a plurality of first and second primers.

In some embodiments, the first and second primers comprise the same sequence. In some embodiments, the first and second primers comprise different sequences.

In some embodiments, the support comprises beads, particles, planar surfaces, or the inner walls of a trough or tube.

In some embodiments, the reaction mixture further comprises a polymerase and a plurality of nucleotides.

In some embodiments, the present disclosure relates generally to compositions comprising emulsions. Optionally, the emulsion comprises a hydrophilic phase and a hydrophobic phase. Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the hydrophilic phase may comprise any combination of a plurality of polynucleotide templates, a plurality of supports, and/or a recombinase. Optionally, the hydrophilic phase may comprise a plurality of polynucleotide templates. Optionally, the hydrophilic phase may comprise a plurality of supports. Alternatively, the hydrophilic phase may comprise a recombinase enzyme.

In some embodiments, the composition comprises an emulsion comprising a hydrophilic phase and a hydrophobic phase, wherein the hydrophilic phase comprises a plurality of polynucleotide templates, a plurality of supports, and a recombinase enzyme.

In some embodiments, the present disclosure generally relates to compositions comprising an emulsion comprising a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the hydrophilic phase comprises a plurality of microreactors. Optionally, at least two microreactors of the plurality comprise different polynucleotide templates. Optionally, the sequences of the different polynucleotide templates are the same or different. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences. Optionally, at least two microreactors of the plurality comprise a recombinase.

In some embodiments, the composition comprises an emulsion comprising a hydrophilic phase dispersed in a hydrophobic phase, wherein the hydrophilic phase comprises a plurality of microreactors, at least two microreactors of the plurality comprising different polynucleotide templates and recombinases.

In some embodiments, the hydrophilic phase comprises a plurality of aqueous microreactors, at least two of which each comprise a different polynucleotide template, support, and recombinase enzyme.

Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences.

In some embodiments, the first and second primers comprise the same sequence.

In some embodiments, the first and second primers comprise different sequences. In some embodiments, the hydrophilic phase further comprises a polymerase.

In some embodiments, the polymerase comprises a strand displacement polymerase. In some embodiments, the hydrophilic phase comprises nucleotides.

In some embodiments, the present disclosure relates generally to methods (and related compositions and systems) for nucleic acid synthesis, comprising: (a) forming a reaction mixture; and (b) subjecting the reaction mixture to amplification conditions. Optionally, the reaction mixture is contained within the hydrophilic phase of the emulsion. Optionally, the emulsion comprises a hydrophilic phase and a hydrophobic phase. Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the reaction mixture comprises any combination of a plurality of supports, a plurality of different polynucleotides, and/or recombinases. Optionally, the reaction mixture comprises a plurality of supports. Optionally, the reaction mixture comprises a plurality of different polynucleotides. Optionally, the sequences of the different polynucleotide templates are the same or different. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences. Optionally, the reaction mixture comprises a recombinase. Optionally, the amplification conditions include isothermal or thermocycling temperature conditions. Optionally, the method further comprises forming at least two supports, subjecting the emulsion to amplification conditions resulting in the formation of a plurality of supports, wherein at least two of the supports are each independently linked to a substantially monoclonal nucleic acid population.

In some embodiments, the present disclosure relates generally to methods (and related compositions and systems) for nucleic acid synthesis, comprising: (a) Forming a reaction mixture comprising a plurality of supports, a plurality of different polynucleotides, and a recombinase enzyme, the reaction mixture being contained within a hydrophilic phase of the emulsion; and (b) subjecting the emulsion comprising the reaction mixture to isothermal amplification conditions, thereby producing a plurality of supports and a population of substantially monoclonal nucleic acids attached thereto.

In some embodiments, the emulsion comprises a water-in-oil emulsion. In some embodiments, the liquid phase microreactor comprises a hydrophilic phase. In some embodiments, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. In some embodiments, the reaction mixture is formed in a single reaction vessel. Optionally, the sequences of a plurality of different polynucleotide templates are the same or different. Optionally, the first polynucleotide template comprises a first sequence and the second polynucleotide template comprises a second sequence. Optionally, the first and second polynucleotide template sequences are the same or different. Optionally, at least one of the plurality of supports is linked to a plurality of first primers (e.g., forward amplification primers). Optionally, the reaction mixture further comprises a plurality of second primers (e.g., reverse amplification primers). In some embodiments, at least one of the plurality of supports further comprises a plurality of second primers. In some embodiments, at least one of the plurality of supports comprises a plurality of first and second primers. In some embodiments, the first and second primers comprise the same sequence. In some embodiments, the first and second primers comprise different sequences. In some embodiments, the nucleic acid synthesis method further comprises recovering at least some of the support attached to the substantially nucleic acid monoclonal population from the reaction mixture. In some embodiments, the nucleic acid synthesis method further comprises placing at least some of the support attached to the substantially monoclonal nucleic acid population on a surface. In some embodiments, the nucleic acid synthesis method further comprises forming an array by placing at least some of the support attached to the substantially monoclonal nucleic acid population on a surface. In some embodiments, the nucleic acid synthesis method further comprises sequencing at least one substantially monoclonal nucleic acid population attached to the support. In some embodiments, the support comprises beads, particles, planar surfaces, or the inner walls of a trough or tube. In some embodiments, the reaction mixture further comprises a polymerase and a plurality of nucleotides. In some embodiments, the polymerase comprises a strand displacement polymerase.

In some embodiments, the method for nucleic acid synthesis comprises forming an emulsion. Optionally, the emulsion comprises a hydrophilic phase and a hydrophobic phase. Optionally, the emulsion comprises a hydrophilic phase dispersed in a hydrophobic phase. Optionally, the hydrophilic phase comprises a plurality of microreactors. Optionally, at least two microreactors in the plurality comprise an individual polynucleotide template. Optionally, at least two microreactors of the plurality comprise different polynucleotide templates. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates have the same or different sequences. Optionally, at least two microreactors of the plurality comprise a recombinase.

In some embodiments, the present disclosure relates generally to methods (and related compositions and systems) for nucleic acid synthesis, comprising: an emulsion is formed comprising a hydrophilic phase dispersed in a hydrophobic phase, the hydrophilic phase comprising a plurality of microreactors, at least two microreactors of the plurality comprising different polynucleotide templates and recombinases.

In some embodiments, the emulsion comprises a water-in-oil emulsion. In some embodiments, the hydrophilic phase further comprises a polymerase. In some embodiments, the polymerase is a strand displacement polymerase. In some embodiments, the hydrophilic phase comprises nucleotides. In some embodiments, the emulsion is formed in a single reaction vessel. Optionally, the sequences of the different polynucleotide templates are the same or different. Optionally, the first microreactor comprises a first polynucleotide template and the second microreactor comprises a second polynucleotide template. Optionally, the first and second polynucleotide templates comprise the same or different sequences. In some embodiments, at least two microreactors in the plurality comprise a plurality of supports. Optionally, at least one of the plurality of supports is linked to a plurality of first primers (e.g., forward amplification primers). Optionally, the reaction mixture further comprises a plurality of second primers (e.g., reverse amplification primers). In some embodiments, at least one of the plurality of supports further comprises a plurality of second primers. In some embodiments, at least one of the plurality of supports comprises a plurality of first and second primers. In some embodiments, the first and second primers comprise the same sequence. In some embodiments, the first and second primers comprise different sequences. In some embodiments, the hydrophilic phase comprises a reaction mixture. In some embodiments, the reaction mixture comprises a plurality of polynucleotide templates, a plurality of supports, and a recombinase. In some embodiments, the method for nucleic acid synthesis further comprises subjecting the emulsion (e.g., comprising the reaction mixture) to isothermal amplification conditions, thereby producing a plurality of substantially monoclonal nucleic acid populations. In some embodiments, a plurality of substantially monoclonal nucleic acid populations are attached to a plurality of supports. In some embodiments, the nucleic acid synthesis method further comprises recovering at least some of the support attached to the substantially nucleic acid monoclonal population from the reaction mixture. In some embodiments, the nucleic acid synthesis method further comprises placing at least some of the support attached to the substantially monoclonal nucleic acid population on a surface. In some embodiments, the nucleic acid synthesis method further comprises forming an array by placing at least some of the support attached to the substantially monoclonal nucleic acid population on a surface. In some embodiments, the nucleic acid synthesis method further comprises sequencing at least one substantially monoclonal nucleic acid population attached to the support. In some embodiments, the support comprises beads, particles, planar surfaces, or the inner walls of a trough or tube. In some embodiments, the reaction mixture further comprises a polymerase and a plurality of nucleotides. In some embodiments, the polymerase comprises a strand displacement polymerase.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. When the definition of a term in an incorporated reference appears to be different from the definition provided in the present teachings, the definition provided in the present teachings shall control.

It will be appreciated that there is an implicit "about" prior to the temperatures, concentrations, times, etc. discussed in the present teachings, and thus slight and insubstantial differences are within the scope of the present teachings.

Unless the context requires otherwise, singular terms will include the plural and plural terms will include the singular.

The terms "comprising," "including," "having," "with," and "having" are not intended to be limiting.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Unless defined otherwise, scientific and technical terms used in connection with the present teachings described herein will have the meaning commonly understood by one of ordinary skill in the art. Generally, nomenclature used in connection with cell and tissue culture, molecular biology, and protein and oligo-or poly-nucleotide chemistry and hybridization, and techniques thereof, are well known and commonly used in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions or as commonly accomplished in the art or as described herein. The techniques and methods described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, e.g., sambrook et al, molecular Cloning: A Laboratory Manual (Third ed., cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. 2000). The nomenclature used in connection with the laboratory procedures and techniques described herein are those well known and commonly employed in the art.

As used in accordance with the exemplary embodiments provided herein, the following terms, unless otherwise indicated, should be understood to have the following meanings:

as used herein, the term "amplifying" and variants thereof include any process for producing multiple copies or complements of at least some portion of a polynucleotide, which is commonly referred to as a "template". The template polynucleotide may be single-stranded or double-stranded. Amplification of a given template may result in the production of a population of polynucleotide amplification products, collectively referred to as "amplicons. The polynucleotide of the amplicon may be single-stranded or double-stranded or a mixture of both. Typically, the template will comprise a target sequence, and the amplicon produced will comprise a polynucleotide having a sequence that is substantially identical or substantially complementary to the target sequence. In some embodiments, the polynucleotides of a particular amplicon are substantially identical or substantially complementary to each other; alternatively, in some embodiments, the polynucleotides within a given amplicon may have nucleotide sequences that differ from one another. Amplification may be performed in a linear or exponential fashion, and may involve repeated and sequential replication of a given template to form two or more amplification products. Some typical amplification reactions include successive and repeated cycles of template-based nucleic acid synthesis, resulting in the formation of a plurality of sub-polynucleotides that comprise at least some portion of the nucleotide sequence of the template and share at least some degree of nucleotide sequence identity (or complementarity) with the template. In some embodiments, each nucleic acid synthesis (which may be referred to as a "cycle" of amplification) includes primer annealing and primer extension steps; optionally, an additional denaturation step may also be included in which the template is partially or completely denatured. In some embodiments, one amplification round comprises a given number of repetitions of a single amplification cycle. For example, an amplification round may include 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or more repetitions of a particular cycle. In one exemplary embodiment, amplification includes any reaction in which a particular polynucleotide template undergoes two successive cycles of nucleic acid synthesis. Synthesis may include template-dependent nucleic acid synthesis. Each cycle of nucleic acid synthesis optionally includes a single primer annealing step and a single extension step. In some embodiments, amplifying comprises isothermal amplification.

As used herein, the term "contacting" and variants thereof, when used with respect to any set of components, includes any process in which the components to be contacted are mixed into the same mixture (e.g., added to the same compartment or solution), and does not necessarily require actual physical contact between the components. The components may be contacted in any order or in any combination (or sub-combination), and may include the following: wherein one or some of said components are subsequently removed from the mixture, optionally before the addition of the other of said components. For example, "contacting a with B and C" includes any and all of the following cases: (i) mixing a with C, followed by adding B to the mixture; (ii) mixing a and B into the mixture; removing B from the mixture, and subsequently adding C to the mixture; and (iii) adding A to a mixture of B and C. "contacting the template with the reaction mixture" includes any or all of the following: (i) Contacting a template with a first component of a reaction mixture to produce a mixture; the other components of the reaction mixture are then added to the mixture in any order or combination; and (ii) the reaction mixture has been fully formed prior to mixing with the template.

As used herein, the term "support" and variants thereof include any solid or semi-solid article upon which an agent, such as a nucleic acid, may be immobilized.

As used herein, the term "isothermal" and variants thereof, when used in reference to any reaction conditions, processes, or methods, includes conditions, processes, and methods that operate under substantially isothermal conditions. Substantially isothermal conditions include any condition in which the temperature is limited to a limited range. In exemplary embodiments, the temperature does not vary by more than 20 ℃, typically not more than 10 ℃, 5 ℃ or 2 ℃. Isothermal amplification includes any amplification reaction in which at least two successive nucleic acid synthesis cycles are performed under substantially isothermal conditions, and includes amplification reactions in which the temperature does not vary by more than 20 ℃, 10 ℃, 5 ℃ or 2 ℃ during the duration of at least two successive nucleic acid synthesis cycles, although the temperature may vary by more than 20 ℃ during other portions of the amplification process, including during other nucleic acid synthesis cycles. Optionally, in an isothermal reaction (including isothermal amplification), the temperature is maintained at or about 50 ℃, 55 ℃, 60 ℃, 65 ℃ or 70 ℃ for at least about 10, 15, 20, 30, 45, 60 or 120 minutes. Optionally, any temperature change does not exceed 20 ℃, optionally within 10 ℃, e.g., within 5 ℃ or 2 ℃, during one or more amplification cycles (e.g., 1, 5, 10, 20 or all amplification cycles performed). In some embodiments, isothermal amplification may include thermal cycling, wherein the temperature change is within an isothermal range. In an example, the temperature change between the denaturation step and another step, such as annealing and/or extension, is limited. In an example, the difference between the denaturation temperature and the annealing or extension temperature does not exceed 20 ℃, optionally within 10 ℃, for example within 5 ℃ or 2 ℃, for one or more amplification cycles. Optionally, temperature variation is limited for at least 5, 10, 15, 20, 30, 35 or substantially all cycles of amplification.

As used herein, the term "sequencing" and variants thereof include obtaining sequence information from a nucleic acid strand, typically by determining information of at least some nucleotides (including nucleobase components thereof) within the nucleic acid molecule. While in some embodiments, "sequencing" a given region of a nucleic acid molecule includes identifying each nucleotide within the region being sequenced, "sequencing" may also include methods in which information for one or more nucleotides is determined while information for some nucleotides remains undetermined or incorrectly determined.

As used herein, the terms "identity" and "identical" and variants thereof, when used with respect to two or more nucleic acid sequences, refer to sequence similarity of two or more sequences (e.g., nucleotide or polypeptide sequences). In the context of two or more homologous sequences, the percent identity or homology of a sequence or subsequence thereof means the ratio of all identical monomer units (e.g., nucleotides or amino acids) (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 95% or 99% identity). When comparing and aligning the maximum correspondence over a window or designated region, as measured by using BLAST or BLAST2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection, the percent identity may be the percent identity over the particular region. Sequences are considered "substantially identical" when there is at least 85% identity at the amino acid level or at the nucleotide level. Preferably, identity exists over a region of at least about 25, 50 or 100 residues in length, or over the entire length of at least one of the sequences being compared. Typical algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST2.0 algorithms, which are described in Altschul et al, nuc. Acids Res.25:3389-3402 (1977). Other methods include the algorithms of Smith & Waterman, adv. Appl. Math.2:482 (1981) and Needleman & Wunsch, J. Mol. Biol.48:443 (1970), etc. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent hybridization conditions.

As used herein with respect to two or more polynucleotides, the term "complementary" and variants thereof refers to polynucleotides comprising any nucleic acid sequence that can undergo cumulative base pairing (as in a hybridized duplex) in an antiparallel direction at two or more individual corresponding sites. Optionally, there may be "complete" or "whole" complementarity between the first and second nucleic acid sequences, wherein each nucleotide in the first nucleic acid sequence may undergo stable base pairing interactions with the polynucleotide in a corresponding antiparallel position on the second nucleic acid sequence (however, the term "complementary" may itself include nucleic acid sequences that are not completely complementary over the entire length); "partial" complementarity describes a nucleic acid sequence in which at least 20% but less than 100% of the residues of the nucleic acid sequence are complementary to residues in other nucleic acid sequences. In some embodiments, at least 50% but less than 100% of the residues of a nucleic acid sequence are complementary to residues in other nucleic acid sequences. In some embodiments, at least 70%, 80%, 90% or 95% but less than 100% of the residues of the nucleic acid sequence are complementary to residues in other nucleic acid sequences. A sequence is considered "substantially complementary" when at least 85% of the residues in one nucleic acid sequence are complementary to residues in other nucleic acid sequences. "non-complementary" describes a nucleic acid sequence in which less than 20% of the residues of the nucleic acid sequence are complementary to residues in other nucleic acid sequences. A "mismatch" exists at any position where two opposing nucleotides are not complementary. Complementary nucleotides include nucleotides that are effectively incorporated relative to each other by a DNA polymerase during DNA replication under physiological conditions. In typical embodiments, complementary nucleotides can form base pairs with each other, such as A-T/U and G-C base pairs formed by specific Watson-Crick type hydrogen bonding between nucleotides and/or nucleobases of the polynucleotide at positions antiparallel to each other. Other artificial base pair complementarity may be based on other types of hydrogen bonding and/or hydrophobic phases of bases and/or shape complementarity between bases.

As used herein with respect to any polynucleotide or nucleic acid molecule, the term "double-stranded" and variants thereof refers to any polynucleotide or nucleic acid molecule having one or more strands and including regions comprising nucleotide residues that base pair with nucleotide residues (e.g., as in a nucleic acid duplex). Optionally, a double-stranded polynucleotide (or nucleic acid molecule) may be "fully" or "entirely" double-stranded, such that each nucleotide residue in the polynucleotide (or nucleic acid molecule) base pairs with another corresponding nucleotide residue. In some embodiments, the double-stranded polynucleotide comprises one or more single-stranded regions comprising nucleotide residues that are not base paired with any other nucleotide residues. In some embodiments, at least 51%, 75%, 85%, 95%, 97%, or 99% of the nucleotide residues in a double-stranded polynucleotide (or nucleic acid molecule) base pair with other nucleotide residues. In some embodiments, a double-stranded polynucleotide (or nucleic acid molecule) comprises two strands that are not covalently linked to each other; alternatively, a double-stranded polynucleotide (or nucleic acid molecule) comprises a single strand that base pairs with itself (e.g., as in a hairpin oligonucleotide) over at least some portion of its length. A polynucleotide is considered "substantially double-stranded" when at least 85% of its nucleotide residues are base-paired with the corresponding nucleotide residues. A nucleic acid sequence is considered "double-stranded" when the residues of the two sequences are base-paired with corresponding residues in the other sequence. In some embodiments, base pairing can occur according to some conventional pairing paradigm, such as A-T/U and G-C base pairs formed by specific Watson-Crick type hydrogen bonding between nucleobases at nucleotide and/or polynucleotide positions antiparallel to each other; in other embodiments, base pairing can occur through any other paradigm in which base pairing occurs according to established and predictable rules.

As used herein, the term "single stranded" and variants thereof, when used with respect to any polynucleotide or nucleic acid molecule, refers to any polynucleotide or nucleic acid molecule that includes a region comprising nucleotide residues that are not base paired with any nucleotide residues. Optionally, a single stranded polynucleotide (or nucleic acid molecule) may be "fully" or "entirely" single stranded, such that each nucleotide residue in the polynucleotide (or nucleic acid molecule) is not base paired with any other nucleotide residue. In some embodiments, a single stranded polynucleotide comprises one or more double stranded regions comprising nucleotide residues that base pair with nucleotide residues. In some embodiments, at least 51%, 75%, 85%, 95%, 97%, or 99% of the nucleotide residues in a single stranded polynucleotide (or nucleic acid molecule) are not base paired with other nucleotide residues. A polynucleotide is considered "substantially single-stranded" when at least 85% of its nucleotide residues are not base-paired with nucleotide residues.

As used herein, the term "denatured" and variants thereof, when used with respect to any double-stranded polynucleotide molecule or double-stranded polynucleotide sequence, includes any process in which base pairing between nucleotides within opposite strands of the double-stranded molecule or double-stranded sequence is disrupted. Typically, denaturation involves rendering at least some portion or region of both strands of a double-stranded polynucleotide molecule or sequence single-stranded. In some embodiments, denaturing comprises separating at least some portion or region of the two strands of a double-stranded polynucleotide molecule or sequence from each other. Typically, the denatured region or portion is then able to hybridize to another polynucleotide molecule or sequence. Optionally, there may be "complete" or "whole" denaturation of the double-stranded polynucleotide molecule or sequence. The complete denaturing conditions are, for example, conditions that result in complete separation of a significant portion (e.g., more than 10%, 20%, 30%, 40%, or 50%) of a large number of strands from their extended and/or full-length complements. Typically, complete or complete denaturation breaks all base pairing of the nucleotides of the two strands with each other. Similarly, a nucleic acid sample is considered completely denatured, optionally when more than 80% or 90% of the individual molecules of the sample lack any double-stranded (or lack any hybridization to complementary strands).

Alternatively, the double stranded polynucleotide molecule or sequence may be partially or incompletely denatured. A given nucleic acid molecule may be considered partially denatured when a portion of at least one strand of the nucleic acid remains hybridized to the complementary strand while another portion is in an unhybridized state (even in the presence of the complementary strand). The unhybridized portion optionally has a length of at least 5, 7, 8, 10, 12, 15, 17, 20, or 50 nucleotides. The hybridized portion optionally has a length of at least 5, 7, 8, 10, 12, 15, 17, 20, or 50 nucleotides. Partial denaturation includes the situation in which some, but not all, of the nucleotides of one strand or sequence are base paired with some of the nucleotides of the other strand or sequence within the double-stranded polynucleotide. In some embodiments, at least 20% but less than 100% of the nucleotide residues of one strand of the partially denatured polynucleotide (or sequence) are not base paired with nucleotide residues within the opposite strand. Under exemplary conditions, at least 50% of the nucleotide residues within a double-stranded polynucleotide molecule (or double-stranded polynucleotide sequence) are in single-stranded (or unhybridized) form, but less than 20% or 10% of the residues are double-stranded.

Optionally, a nucleic acid sample is considered partially denatured when a substantial portion (e.g., more than 20%, 30%, 50%, or 70%) of the individual nucleic acid molecules of the sample are in a partially hybridized state. Optionally, less than a substantial amount of the individual nucleic acid molecules in the sample are completely denatured, e.g., no more than 5%, 10%, 20%, 30% or 50% of the nucleic acid molecules in the sample. Under exemplary conditions, at least 50% of the sample's nucleotide molecules are partially denatured, but less than 20% or 10% are fully denatured. In other cases, at least 30% of the sample's nucleotide molecules are partially denatured, but less than 10% or 5% are fully denatured. Similarly, a nucleic acid sample may be considered non-denatured when a minority of the individual nucleic acid molecules in the sample are partially or completely denatured.

In embodiments, the partial denaturation conditions are achieved by maintaining the duplex in an appropriate temperature range. For example, the nucleic acid is maintained at a temperature that is sufficiently elevated to achieve some thermal denaturation (e.g., above 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, or 70 ℃) but not sufficiently high to achieve complete thermal denaturation (e.g., below 95 ℃ or 90 ℃ or 85 ℃ or 80 ℃ or 75 ℃). In embodiments, the nucleic acid is partially denatured using substantially isothermal conditions.

Partial denaturation can also be achieved by other methods, for example chemical methods using a chemical denaturant such as urea or formamide in a concentration appropriately adjusted or using a high or low pH (e.g. a pH between 4-6 or 8-9). In embodiments, partial denaturation and amplification is achieved using recombinase-polymerase amplification (RPA). Exemplary RPA methods are described herein.

In some embodiments, complete or partial denaturation is achieved by treating the double stranded polynucleotide sequence to be denatured with an appropriate denaturing agent. For example, a double-stranded polynucleotide may be subjected to thermal denaturation (also interchangeably referred to as thermal denaturation) by raising the temperature to a point where a desired level of denaturation is achieved. In some embodiments, the temperature may be adjusted to achieve complete separation of the two strands of the polynucleotide, such that at least 90% of the strands are in single stranded form throughout their length. In some embodiments, complete thermal denaturation of a polynucleotide molecule (or polynucleotide sequence) is achieved by exposing the polynucleotide molecule (or sequence) to a temperature at least 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25, 30 ℃, 50 ℃, or 100 ℃ above the calculated or predicted melting temperature (Tm) of the polynucleotide molecule or sequence.

Alternatively, chemical denaturation can be achieved by contacting the double stranded polynucleotide to be denatured with a suitable chemical denaturant, such as a strong base, strong acid, a pro-solvent, etc., and may include, for example, naOH, urea, or guanidine-containing compounds. In some embodiments, partial or complete denaturation is achieved by exposure to a chemical denaturant such as urea or formamide at a concentration appropriately adjusted or using a high or low pH (e.g., a pH between 4-6 or 8-9). In embodiments, partial denaturation and amplification is achieved using recombinase-polymerase amplification (RPA). Exemplary RPA methods are described herein.

The terms "melting temperature", "Tm" or "T", when used with respect to a given polynucleotide (or a given target sequence within a polynucleotide) _m "and variants thereof generally refer to a temperature at which 50% of a given polynucleotide (or a given target sequence) exists in double-stranded form and 50% is single-stranded under a defined set of conditions. In some embodiments, the set of determined conditions may include a determined parameter indicative of ionic strength and/or pH in the aqueous reaction conditions. The determined conditions can be adjusted by varying the concentration of salts (e.g., sodium), temperature, pH, buffers, and/or formamide. Typically, the calculated thermal melting temperature may be T _m Below about 5-30 ℃ or T _m Below about 5-25 ℃ or T _m Below about 5-20 ℃ or T _m Below about 5-15 ℃ or T _m About 5-10 c below. For calculating T _m Methods of (2) are well known and can be found in Sambrook (1989in"Molecular Cloning:A Laboratory Manual", 2) ^nd edition, volumes1-3; wetmur 1966, J.mol. Biol.,31:349-370; wetmur 1991Critical Reviews in Biochemistry and Molecular Biology,26:227-259). For calculating T for hybridization or denaturation of nucleic acids _m Other sources of (a) include OligoAnalyze (from Integrated DNA Technologies) and Primer3 (published by the Whitehead Institute for Biomedical Research). In some embodiments, the terms "melting temperature", "Tm" and "T _m "and variants thereof include a given polynucleotideThe actual Tm (as measured empirically using established conditions) or predicted or calculated Tm of (or target sequence). In some embodiments, the Tm of a template can be predicted or calculated without using the sequence of the template by assuming that the template comprises a proportion of 4 common nucleotides (A, C, G and T) and has a length (or average length in the case of a population of templates). For example, it may be assumed that a population of templates migrating tailing on a gel contains 25% of each A, C, G or T, and has an average length of 200, 300, 400 base pairs.

As used herein with respect to a chemical moiety, the term "label" and variants thereof include any composition comprising an optically or non-optically detectable moiety, wherein the detectable moiety has been manually added, linked or attached to a second moiety that is not labeled by chemical treatment. Typically, the user (or upstream provider) adds a marking for the purpose of enhancing the detectability of the second portion. Optically or non-optically detectable components of the composition that are already present in the naturally occurring form of the composition (e.g., hydrogen ions and amino acids present in typical DNA molecules, RNA molecules, or nucleotides within a natural cell) are not labels for the purposes of the present disclosure. Some typical labels include fluorescent moieties and dyes.

A nucleic acid may be considered immobilized if it is attached to the support in a substantially stable manner at least during selected conditions (e.g., during an amplification reaction). The attachment may be by any mechanism including, but not limited to, non-covalent bonding, ionic interactions, covalent attachment. A first nucleic acid is also considered to be immobilized to a support during amplification if the conditions of amplification are such that substantial amounts of the first and second nucleic acids are associated or linked to each other at any or all times during amplification. For example, the first and second nucleic acids can be associated together by hybridization including Watson-Crick base pairing or hydrogen bonding. In examples, the amplification conditions selected allow at least 50%, 80%, 90%, 95% or 99% of the first nucleic acid to remain hybridized to the second nucleic acid, or vice versa. Nucleic acid may be considered to be non-immobilized or non-immobilized if it is not directly or indirectly attached or associated with a support.

A vehicle may be considered to be flowable under selected conditions if it is at least temporarily a fluid vehicle that does not substantially or completely inhibit or impede the transfer or movement of the non-immobilized molecules under the selected conditions. The non-immobilized molecule is not itself immobilized to a solid support or surface or is not associated with another immobilized molecule. In embodiments, the non-immobilized molecule is a solute (e.g., a nucleic acid) that passes through a flowable vehicle. The transfer or movement in an exemplary vehicle may be from any first point in the continuous phase to any other point in the continuous phase that is in fluid communication or the same continuous phase by diffusion, convection, turbulence, agitation, brownian motion, advection, electric current, or other molecular motion within the liquid. For example, in a flowable vehicle, a significant amount of non-immobilized nucleic acid is transferred from one immobilization site to another immobilization site within the same continuous phase of the flowable vehicle or in fluid communication with the first immobilization site. Optionally, the rate of transfer or movement of nucleic acid in the vehicle is comparable to the rate of transfer or movement of nucleic acid in water. In some examples, the conditions selected are conditions experienced by the vehicle during amplification. The selected conditions may or may not allow the flowable vehicle to remain substantially stationary. The conditions may or may not subject the flowable vehicle to efficient mixing, agitation, or shaking. The vehicle is optionally flowable at least temporarily during amplification. For example, the vehicle is flowable under the at least one pre-amplification and/or amplification conditions selected. Optionally, the flowable vehicle does not substantially prevent mixing of different non-immobilized nucleic acids or transfer of non-immobilized nucleic acids between different regions of the continuous phase of the flowable vehicle. The movement or transfer of nucleic acids may be caused, for example, by diffusion or convection. The vehicle is optionally considered to be non-flowable if the non-immobilized nucleic acid cannot propagate or migrate between different immobilization sites or throughout the continuous phase after amplification. Generally, the flowable vehicle does not substantially confine the non-immobilized nucleic acids (e.g., templates or amplicons) within the effective area of the reaction volume or to a fixed location during the amplification period. Optionally, the flowable vehicle can be rendered non-flowable by a variety of methods or by changing the conditions of the flowable vehicle. Optionally, if the vehicle is liquid or not semi-solid, it is flowable. The vehicle may be considered flowable if its fluidity is comparable to pure water. In other embodiments, a vehicle may be considered flowable if it is a fluid that is substantially free of polymer, or if its viscosity coefficient is similar to that of pure water.

As will be appreciated by those of skill in the art, references to templates, starter oligonucleotides, extension probes, primers, etc., may refer to groups or libraries of nucleic acid molecules that are substantially identical within the relevant portion, rather than individual molecules. For example, a "template" may refer to a plurality of substantially identical template molecules; "probe" may refer to a plurality of substantially identical probe molecules, and the like. In the case of probes that are degenerate in one or more positions, it will be appreciated that the sequences of the probe molecules comprising a particular probe will differ in degenerate positions, i.e., the sequences of the probe molecules comprising a particular probe may be substantially identical only in non-degenerate positions. These terms within the present application are intended to provide support for a population or molecule. When intended to refer to a single nucleic acid molecule (i.e., one molecule), the terms "template molecule," "probe molecule," "primer molecule," and the like may be used interchangeably. In certain examples, the plural nature of substantially identical groups of nucleic acid molecules will be explicitly indicated.

"template," "oligonucleotide," "probe," "primer," "template," "nucleic acid," and the like are intended herein to be interchangeable terms. These terms refer to polynucleotides, which are not necessarily limited to any length or function. The same nucleic acid may be regarded as a "template", "probe" or "primer" depending on the context, and may switch between these roles over time. "Polynucleotide" is also referred to as "nucleic acid" which is a linear polymer of two or more nucleotides, or variants or functional fragments thereof, linked by covalent internucleoside linkages. In naturally occurring examples of these, the internucleoside linkage is typically a phosphodiester linkage. However, other examples optionally include other internucleoside linkages, such as phosphorothioate (phosphorothioate) linkages, and may or may not include phosphate groups. Polynucleotides include double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA, DNA: RNA hybrids, peptide Nucleic Acids (PNAs) and hybrids between PNA and DNA or RNA, and may also include known types of modifications. The polynucleotides may optionally be linked via the 5 'or 3' end to one or more non-nucleotide moieties such as labels and other small molecules, macromolecules such as proteins, lipids, sugars and solid or semi-solid supports. Labels include any moiety that can be detected using a selected detection method and thus make the linked nucleotide or polynucleotide similarly detectable using a selected detection method. Optionally, the marker emits optically detectable or visible electromagnetic radiation. In some cases, the nucleotide or polynucleotide is not attached to a label, but the presence of the nucleotide or polynucleotide is detected directly. "nucleotide" refers to a nucleotide, nucleoside or analog thereof. Optionally, the nucleotide is an N-or C-glycoside of a purine or pyrimidine base. (e.g., deoxyribonucleosides comprising 2-deoxy-D-ribose or ribonucleosides comprising D-ribose). Examples of other analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides. Reference to a nucleic acid by any of these terms should not be construed to mean that the nucleic acid has any particular activity, function or property. For example, the word "template" does not mean that the "template" is being replicated by a polymerase or that the template cannot be used as a "primer" or "probe".

It will be appreciated that in certain examples, nucleic acid reagents, such as templates, probes, primers, etc., involved in amplification may be part of a larger nucleic acid molecule that also comprises another part that does not have the same function. Optionally, the other moiety does not have any template, probe or primer function. In some examples, a nucleic acid that substantially hybridizes to an optionally immobilized primer (e.g., on an immobilization site) is considered a "template". Any one or more nucleic acid reagents (template, immobilized strand, immobilized or non-immobilized primer, etc.) involved in template walking may be generated from other nucleic acids prior to or during amplification. Nucleic acid reagents are optionally generated (and need not be identical) from the input nucleic acid by one or more modifications to the nucleic acid that was originally introduced into the template walking vehicle. The input nucleic acid may, for example, be subjected to restriction digests, ligation, one or more amplification cycles, denaturation, mutation, etc., to produce nucleic acids that serve as templates, primers, etc., during amplification or further amplification. For example, a double-stranded input nucleic acid may be denatured to produce a first single-stranded nucleic acid, which is optionally used to produce a second complementary strand. If so desired, the first single stranded nucleic acid may be considered a "template" for my purposes herein. Alternatively, the second complementary strand generated from the first single-stranded nucleic acid may be considered a "template" for my purposes herein. In another example, the template is derived from and not necessarily identical to the input nucleic acid. For example, the template may comprise additional sequences not present in the input nucleic acid. In embodiments, the template may be an amplicon generated from an input nucleic acid using one or more primers having 5' overhangs that are not complementary to the input nucleic acid.

As used herein with respect to two or more polynucleotides, the term "hybridization" and variants thereof refers to any process in which any one or more nucleic acid sequences (each sequence comprising a segment of contiguous nucleotide residues) within the polynucleotide undergo base pairing (e.g., as in a hybridized nucleic acid duplex) at two or more individual corresponding positions. Optionally, there may be "complete" or "complete" hybridization between the first and second nucleic acid sequences, wherein each nucleotide residue in the first nucleic acid sequence may undergo base pairing interactions with a corresponding nucleotide in an antiparallel position on the second nucleic acid sequence. In some embodiments, hybridization may include base pairing between two or more nucleic acid sequences that are not fully complementary or base-paired over their entire length. For example, a "partial" hybridization occurs when two nucleic acid sequences undergo base pairing, wherein at least 20% but less than 100% of the residues of one nucleic acid sequence are base paired with residues in the other nucleic acid sequence. In some embodiments, hybridization comprises base pairing between two nucleic acid sequences, wherein at least 50% but less than 100% of the residues of one nucleic acid sequence base pair with corresponding residues in the other nucleic acid sequence. In some embodiments, at least 70%, 80%, 90% or 95% but less than 100% of the residues of one nucleic acid sequence base pair with corresponding residues in other nucleic acid sequences. Two nucleic acid sequences are considered to be "substantially hybridized" when at least 85% of the residues of one nucleic acid sequence are base paired with corresponding residues in the other nucleic acid sequence. In the case where one nucleic acid molecule is substantially longer than the other (or where two nucleic acid molecules comprise regions that are substantially complementary and substantially non-complementary), the two nucleic acid molecules may be described as "hybridized" even when portions of one or both nucleic acid molecules may remain unhybridized. "unhybridized" describes a nucleic acid sequence in which less than 20% of the residues of one nucleic acid sequence base pair with residues in the other nucleic acid sequence. In some embodiments, base pairing can occur according to some conventional pairing paradigm, such as A-T/U and G-C base pairs formed by specific Watson-Crick type hydrogen bonding between nucleobases at nucleotide and/or polynucleotide positions antiparallel to each other; in other embodiments, base pairing can occur through any other paradigm in which base pairing occurs according to established and predictable rules.

Hybridization of two or more polynucleotides can occur whenever the two or more polynucleotides are contacted under appropriate hybridization conditions. Hybridization conditions include any conditions suitable for hybridization of nucleic acids; methods for performing hybridization and suitable conditions for hybridization are well known in the art. The stringency of hybridization can be affected by a number of parameters, including the degree of identity and/or complementarity between the polynucleotides to be hybridized (or any target sequences within the polynucleotides); the melting temperature of the polynucleotide and/or target sequence to be hybridized, which is referred to as "T", is _m "; parameters such as salt, buffer, pH, temperature, GC% content of polynucleotide and primer and/or time. Typically, hybridization is at a lower levelIs advantageous at elevated temperatures and/or elevated salt concentrations and reduced organic solvent concentrations. High stringency hybridization conditions will generally require a higher degree of complementarity between the two target sequences for hybridization to occur, while low stringency hybridization conditions will promote hybridization even when the two polynucleotides to be hybridized exhibit a low level of complementarity. Hybridization conditions may be applied during the hybridization step or optional and sequential wash steps or hybridization and optional wash steps.

Examples of high stringency hybridization conditions include any one or more of the following: a salt (e.g., naCl) concentration of about 0.0165 to about 0.0330; melting temperature (T) of target sequence (or polynucleotide) to be hybridized _m ) A temperature below about 5 ℃ to about 10 ℃; and/or a formamide concentration of about 50% or greater. Generally, high stringency hybridization conditions allow for binding between sequences having high homology, e.g., > 95% identity or complementarity. In one exemplary embodiment of high stringency hybridization conditions, the compositions comprise 25mM KPO at about 42 DEG C ₄ Hybridization was performed in hybridization solutions of (pH 7.4), 5 XSSC, 5 XDenhardt's solution, 50. Mu.g/mL denatured sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL double-stranded polynucleotide (or double-stranded target sequence), while washing was performed at about 65℃with a wash solution comprising 0.2 XSSC and 0.1% sodium dodecyl sulfate.

Examples of moderately stringent hybridization conditions include any one or more of the following: a salt (e.g., naCl) concentration of about 0.165 to about 0.330; the melting temperature (T) _m ) A temperature below about 20 ℃ to about 29 ℃; and/or a formamide concentration of about 35% or less. Typically, such moderately stringent hybridization conditions allow for binding between sequences having high or moderate homology, e.g., > 80% identity or complementarity. In one exemplary embodiment of moderately stringent hybridization conditions, the hybridization temperature is about 42℃at a temperature comprising 25mM KPO ₄ Hybridization was performed in hybridization solution of (pH 7.4), 5 XSSC, 5 XDenhart solution, 50. Mu.g/mL denatured sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL double-stranded polynucleotide (or double-stranded target sequence), while washing was performed at about 50℃using a washing solution comprising 2 XSSC and 0.1% sodium dodecyl sulfateAnd (5) washing.

Examples of low stringency hybridization conditions include any one or more of the following: a salt (e.g., naCl) concentration of about 0.330 to about 0.825; the melting temperature (T) _m ) A temperature below about 40 ℃ to about 48 ℃; and/or a formamide concentration of about 25% or less. Typically, such low stringency conditions allow for binding between sequences that have low homology, e.g., > 50% identity or complementarity.

Some exemplary conditions suitable for hybridization include incubating the polynucleotides to be hybridized in a solution having a sodium salt, such as NaCl, sodium citrate, and/or sodium phosphate. In some embodiments, the hybridization or wash solution may comprise about 10-75% formamide and/or about 0.01-0.7% Sodium Dodecyl Sulfate (SDS). In some embodiments, the hybridization solution may be a stringent hybridization solution, which may include any combination of 50% formamide, 5X SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt solution, 0.1% SDS, and/or 10% dextran sulfate. In some embodiments, the hybridization or wash solution may comprise BSA (which may be in bovine serum albumin). In some embodiments, hybridization or washing may be performed at a temperature in the range of about 20-25 ℃, or about 25-30 ℃, or about 30-35 ℃, or about 35-40 ℃, or about 40-45 ℃, or about 45-50 ℃, or about 50-55 ℃ or higher.

In some embodiments, hybridization or washing may be performed for a time period ranging from about 1 to 10 minutes, or from about 10 to 20 minutes, or from about 20 to 30 minutes, or from about 30 to 40 minutes, or from about 40 to 50 minutes, or from about 50 to 60 minutes or more.

In some embodiments, hybridization or washing may be performed at a pH in the range of about 5 to 10, or about pH6 to 9, or about pH6.5 to 8, or about pH6.5 to 7.

In some embodiments, the term "monoclonal" and variants thereof are used to describe a population of polynucleotides in which a substantial portion (e.g., at least about 50%, typically at least 75%, 80%, 85%, 90%, 95%, or 99%) of the population members share at least 80% identity at the nucleotide sequence level. Typically, at least about 90%, typically at least about 95%, more typically at least about 99%, 99.5% or 99.9% of the population is produced by amplification or template-dependent replication of a specific polynucleotide sequence present in a substantial portion of a member of the population of monoclonal polynucleotides. All members of a monoclonal population need not be identical or complementary to each other. For example, different portions of a polynucleotide template may be amplified or replicated to produce members of a resulting monoclonal population; similarly, a certain number of "errors" and/or incomplete extensions may occur during amplification of the original template, thereby generating a monoclonal population whose individual members may exhibit sequence differences between themselves. In some embodiments, at least 50% of the members of the monoclonal population are at least 80% identical to a reference nucleic acid sequence (i.e., a nucleic acid having a defined sequence that is used as a basis for sequence comparison). In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more members of the population comprise sequences having at least 80%, 85%, 90%, 95%, 97% or 99% identity (or complementarity) to a reference nucleic acid sequence. In some embodiments, low or insubstantial levels of mixing of non-homologous polynucleotides may occur during the nucleic acid amplification reactions described herein, and thus a substantially monoclonal population may comprise a minority of different polynucleotides (e.g., less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.001% of different polynucleotides). As used herein, the phrase "substantially monoclonal" and variants thereof, when used with respect to one or more polynucleotide populations, refers to one or more polynucleotide populations comprising polynucleotides having at least 80% identity to an original single template used as a basis for clonal amplification to produce a substantially monoclonal population.

In some embodiments, at least 80% of the members of the amplicon, typically at least 90%, more typically at least 95%, more typically at least 99% of the members of the amplicon, will share greater than 90% identity, typically greater than 95% identity, more typically greater than 97% and more typically greater than 99% identity with the polynucleotide template. Alternatively, members of the amplicon may have greater than 90% complementarity, typically greater than 95% complementarity, more typically greater than 97% complementarity, and more typically greater than 99% complementarity to the original template. In some embodiments, members of a substantially monoclonal nucleic acid population can hybridize to each other under stringent hybridization conditions.

In some embodiments, an amplicon is said to be "monoclonal" or "substantially monoclonal" if the amplicon contains sufficiently little polyclonal contaminants to produce a detectable signal in any method of nucleic acid analysis that is affected by the template sequence. For example, a "monoclonal" population of polynucleotides may include any population that produces a signal (e.g., a sequencing signal, a nucleotide incorporation signal, etc.) that can be detected using a particular sequencing system. Optionally, the signal may then be analyzed to correctly determine the sequence and/or base information of any one or more nucleotides present within any polynucleotide of the population. Examples of suitable sequencing systems for detecting and/or analyzing such signals include Ion Torrent sequencing systems, e.g., ion Torrent PGM ^TM Sequencing systems, including 314, 316 and 318 systems, and Ion Torrent Proton ^TM Sequencing systems, including Proton I, proton II and Proton III (Life Technologies, carlsbad, calif.). In some embodiments, the monoclonal amplicon allows for accurate sequencing of at least 5 consecutive nucleotide residues on an Ion Torrent sequencing system.

As used herein, the term "clonal amplification" and variants thereof refers to any process in which a population of substantially monoclonal polynucleotides is produced by amplification of a polynucleotide template. In some embodiments of clonal amplification, two or more polynucleotide templates are amplified to produce at least two substantially monoclonal polynucleotide populations.

As used herein, the term "linker" includes polynucleotides or oligonucleotides comprising DNA, RNA, chimeric RNA/DNA molecules, analogs thereof and generally means an added or foreign sequence that is linked or bound to a target polynucleotide of interest (e.g., a template) during an operational process. Ligation of the linker to the template may optionally occur before or after amplification of the template. In some embodiments, the adapter may comprise a primer binding sequence that is substantially identical or substantially complementary to a sequence within the corresponding primer. In some embodiments, a first adaptor comprising a first primer binding site is attached to one end of a linear double stranded template, and a second adaptor comprising a second primer binding site is attached to the other end.

As used herein, the term "binding partner" includes two molecules or portions thereof that have a specific binding affinity for each other and will generally bind to each other in preference to binding to other molecules. Typically, but not necessarily, some or all of the structure of one member of a particular binding pair is complementary to some or all of the structure possessed by the other member, wherein the two members are capable of binding together, particularly by means of a bond between the complementary structures (optionally by multiple non-covalent attractive forces).

In some embodiments, the molecules used as binding partners include: biotin (and derivatives thereof) and its binding partner avidin moiety, streptavidin moiety (and derivatives thereof); his-tag binding nickel, cobalt or copper; cysteine, histidine or histidine stretch (histidin patch) binding to Ni-NTA; maltose binding to Maltose Binding Protein (MBP); lectin-carbohydrate binding partners; calcium-Calcium Binding Protein (CBP); acetylcholine and receptor-acetylcholine; protein a and a binding partner anti-FLAG antibody; GST and binding partner glutathione; uracil DNA Glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) proteins; an antigen or epitope tag that binds to an antibody or antibody fragment, particularly an antigen such as digoxigenin, fluorescein, dinitrophenol, or bromodeoxyuridine with its respective antibody; mouse immunoglobulin and goat anti-mouse immunoglobulin; bound IgG and protein a; receptor-receptor agonists or receptor antagonists; enzyme-enzyme cofactors; enzyme-enzyme inhibitor; and thyroxine-cortisol. Another binding partner for biotin may be a biotin binding protein from chicken (Hytonen et al, BMC Structural Biology 7:8).

The avidin moiety may include avidin and any derivative of avidin that can bind to the biotin moietyAnalogs and other non-natural forms. Other forms of avidin moieties include native and recombinant avidin and streptavidin, as well as derived molecules such as non-glycosylated avidin, N-acyl avidin, and truncated streptavidin. For example, the avidin moiety includes deglycosylated forms of avidin, bacterial streptavidin produced by Streptomyces (e.g., streptomyces avermitilis (Streptomyces avidinii)), truncated streptavidin, recombinant avidin and streptavidin and derivatives of natural, deglycosylated and recombinant avidin and natural, recombinant and truncated streptavidin, such as N-acyl avidin, e.g., N-acetyl, N-phthaloyl and N-succinyl avidin, and the commercial products ExtrAvidin ^TM 、Captavidin ^TM 、Neutravidin ^TM And Neutralite Avidin ^TM 。

Examples

Embodiments of the present teachings will be further understood from the following examples, which should not be construed as limiting the scope of the present teachings in any way.

Example 1

The nucleic acid amplification reaction was performed in a single continuous liquid phase in a total reaction volume of 220. Mu.L in a single reaction vessel.

About 420x10 with water in a 1.5mL tube (tube 1) ⁶ The beads of (a) were washed 1 time (vortexing/spinning) followed by 1 time (vortexing/spinning) in buffer.

The source of the recombinant enzyme is from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). The dehydrated pellet in the kit comprises usvX recombinase, usvY recombinase loading protein, gp32 protein, bsu DNA polymerase, dNTP, ATP, phosphocreatine, and creatine kinase. Rehydration from TwitAmp in 120. Mu.L of rehydration buffer provided by the kit ^TM Four pellet of Basic kit (tube 2). The recombinase solution was vortexed and spun, followed by ice-cooling. Preparation of two heatsThe blocks, one set at about 68-70 ℃ and one set at 40 ℃.

The supernatant was removed from the pellet (tube 1) leaving about 20. Mu.L of liquid at the bottom.

Reverse primer (2 μl of 100 μΜ stock) was added to the bead tube (tube 1), followed by vortexing and spinning. Reverse primer sequence: 5' -ATCCCTGCGTGTCTCCGAC-3.

Biotinylated reverse primer (2 μl of 10 μΜ stock) was added to the bead tube (tube 1), followed by vortexing and spinning. Biotinylated reverse primer sequence: 5'Bio-ATCCCTGCGTGTCTCCGAC-3'.

mu.L of the polynucleotide library (at different concentrations) was added to the bead tube (tube 1), vortexed/spun, and placed on ice. Library concentrations varied according to the desired DNA to bead ratios of 1:50, 1:75, 1:200.

The rehydrated recombinase mixture (tube 2, reconstituted in 120 μl rehydration buffer) was added to the bead tube (tube 1), vortexed, spun; and placed on ice.

65 μl of the exemplary sieving agent of the present disclosure was added to the bead tube, vortexed and spun, and placed on ice.

mu.L of ice-cold 280mM Mg-acetate was added to the bead tube (in the middle), then vortexed for 3 seconds at maximum setting and put back on ice for 10 seconds and incubated on a heating block for 20 minutes at 40 ℃.

The reaction was heat inactivated in a heating block at 68-70 ℃ for 10 minutes.

The reaction tube was topped up with TE buffer, vortexed and spun for 3 minutes at maximum setting (-20 KG) and the solution was removed from the beads leaving-100. Mu.L. The washing step was repeated twice.

The beads were washed once with the recovery solution.

The reaction tube was topped up with wash buffer, vortexed and spun for 3 minutes at maximum setting (-20 KG), and the solution was removed from the beads leaving-100 μl. The washing step was repeated twice.

After the final spin, the solution was reduced to 100 μl (wash solution).

By conjugating biotinylated polynucleotides to antibioticsParamagnetic beads of streptavidin (MyOne from Dynabeads) ^TM Bead) to enrich the beads.

The enriched beads were loaded into Ion Torrent Ion sensitive chips and subjected to standard sequencing reactions. A significant portion of the enriched beads are determined to comprise a substantially monoclonal population of amplified polynucleotides, e.g., by Ion Torrent PGM ^TM Observations of detectable sequencing signals from such beads on a sequencer were confirmed. The sequencing signal is analyzed to determine the sequence present within the amplicon of each such bead.

Example 2

Will be about 240x10 ⁶ The beads (with forward primer bound) were washed once in an annealing buffer (from Ion Sequencing kit, e.g. PN 4482006) in a 2mL tube. Remove (except 50. Mu.L) and discard the supernatant. The beads were resuspended in 100. Mu.L of annealing buffer.

Will have 300bp or 400bp insert (about 120-240x10 ⁶ Copy) of the barcode DNA library is prehybridized to the washed beads. The library comprises an insert sequence linked at one end to a linker hybridized to a forward primer and at the other end to a linker hybridized to a reverse primer. The template/bead ratios tested included 1:1, 0.75:1, and 0.5:1. The final volume was adjusted to 200. Mu.L with annealing buffer. Through a vortex and spin mixing tube. The tubes were incubated at 95-100℃for 3 min and at 37℃for 5 min. 1mL of annealing buffer was added and the supernatant was discarded at 3.5 minutes above 16,000XG vortex and spin tube. 1mL of 10mM potassium acetate was added, vortexed and spun for 3.5 minutes at above 16,000XG, and the supernatant discarded. The potassium acetate wash was repeated once. The beads were resuspended in 480 μl potassium acetate (tube 1).

The source of the recombinant enzyme is from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). A list of components in the dehydrated precipitate from Basic kit can be found in example 1 above. Rehydration from the TwistAmp in 2.88mL of the rehydration buffer provided by the kit in a 15mL tube (tube 2) ^TM 96 pellets of Basic kit.

mu.L of 100. Mu.M reverse primer (non-immobilized primer) was added to the washed/prehybridized beads (tube 1). 48. Mu.L of 10. Mu.M biotinylated reverse primer (non-immobilized primer) was added to the washed/prehybridized beads and vortexed (tube 1). The contents of tube 1 (containing library, beads and reverse primer) were added to tube 2 (containing rehydrated pellet), tube 2 was vortexed for 5 seconds and placed on ice. 144. Mu. L T4 gp32 protein (15. Mu.g/. Mu.L) was added, vortexed and placed back on ice. 1.56mL of an exemplary sieving agent of the present disclosure was added, swirled and placed back on ice. After the reaction was kept on ice for more than 5 minutes, 264 μl of magnesium acetate was added, vortexing the tube 3 times for 3 seconds each. 50 μl samples were aliquoted into ice-chilled 96-well plates. The 96-well plates were incubated at 40℃for 25 minutes on a thermal cycler (temperature maintained at 40 ℃).

To terminate the reaction, 150 μl of 100mM EDTA was added to each well. All reactions were pooled and centrifuged at above 16,000XG for 3.5 minutes. The supernatant was discarded. 1mL of Tris/1% SDS was added, vortex tube. The beads were washed twice in 1mL OneTouch wash solution. The beads were resuspended in 100 μl.

By interaction with paramagnetic streptavidin beads (MyOne from Dynabeads ^TM Beads) to enrich for the beads that flank copies of the library. The enriched beads were loaded into Ion Torrent PGM Ion sensitive chips.

According to Ion PGM ^TM Standard Sequencing reactions were performed in the Sequencing 400Kit (User Guide PN 4474246B) according to the manufacturer's instructions. A significant portion of the enriched beads loaded onto the chip are determined to comprise a substantially monoclonal population of amplified polynucleotides, such as by Ion Torrent PGM ^TM Observations of detectable sequencing signals from these beads on the sequencer were confirmed. The sequencing signal was analyzed by Torrent Suite Software to determine the sequences present within the amplicons of these beads.

Sequencing data resulted in an average read length of 305bp (FIG. 9), and the mass measurements of the alignment were 1.16G (AQ 17) and 1.07G (AQ 20).

Example 3

Will be about 250x10 ⁶ Bead (combined)Forward primer) was washed once in 1.5mL of annealing buffer (from Ion Sequencing kit, e.g., PN 4482006), vortexed at 15,000xg and spun for 6 minutes. The supernatant was discarded, leaving about 50 μl in the tube.

Will have a 140bp insert (about 50x10 ⁶ Copy) of the library was prehybridized to the washed beads. The library comprises an insert sequence linked at one end to a linker hybridized to a forward primer and at the other end to a linker hybridized to a reverse primer. The library (0.81. Mu.L of 62M stock) and 0.1mL of annealing buffer were added to the washed beads and mixed by pipetting up and down. The bead/template ratio was about 5:1. The tube was incubated at 92-95℃for 7 min and at 37℃for 10 min. 1mL of annealing buffer was added, vortexed and spun for 6 minutes above 15,000XG, and the supernatant discarded. 1mL of 10mM potassium acetate was added, vortexed and spun for 6 minutes above 15,000XG, and the supernatant discarded. The potassium acetate wash was repeated once and the tube was placed on ice. About 60. Mu.L of liquid remained in the tube (tube 1).

The source of the recombinant enzyme is from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). A list of components in the dehydrated precipitate from Basic kit can be found in example 1 above. Rehydrating the samples from the TwistAmp in about 240. Mu.L of rehydration buffer provided by the kit ^TM 8 precipitates from Basic kit. The pellet and rehydration buffer were vortexed, spun and ice-cooled.

mu.L of 100. Mu.M reverse primer (non-immobilized primer) and 1. Mu.L of 10. Mu.M biotinylated reverse primer (non-immobilized primer) were added to the washed/prehybridized beads (tube 1), and the tube (tube 1) was vortexed and spun. The contents of tube 1 (including library, beads and reverse primer) were added to tube 2 (including rehydrated pellet) and tube 2 was vortexed and placed on ice. 130 μl of the exemplary sieving agent of the present disclosure was added, swirled and placed back on ice. 24 μl ice-cold 280mM magnesium acetate was added, vortexed and spun. The total reaction volume was about 332 μl. The tubes were incubated at 40℃for 60 minutes.

1ml of 100mM EDTA was added to terminate the reaction. The tube was vortexed and spun at 15,000XG for 6 minutes. The supernatant was discarded and left in the tube at about 20 μl. The EDTA termination reaction step, vortexing and spinning steps were repeated. 1mL of Tris/1% SDS was added and the tube was vortexed and spun at 15,000XG for 6 minutes. The supernatant was discarded and left in the tube at about 50 μl. The beads were washed by vortexing and spinning in 1mL OneTouch wash solution, leaving about 100 μl in the tube. All reactions were pooled and spun at 15,000XG for 6 minutes, the supernatant discarded, leaving about 100. Mu.L in the tube.

By interaction with paramagnetic streptavidin beads (MyOne from Dynabeads ^TM Beads) to enrich for the beads that flank copies of the library. Loading of enriched beads into Ion Torrent Proton I ^TM Ion sensitive chip.

According to Ion PI ^TM Standard Sequencing reactions were performed according to the manufacturer's instructions in Sequencing 200Kit (User Guide PN MAN 0007491). A significant portion of the enriched beads loaded onto the chip are determined to comprise a substantially monoclonal population of amplified polynucleotides, e.g., by Ion Torrent Proton ^TM Observations of detectable sequencing signals from these beads on the sequencer were confirmed. The sequencing signal was analyzed by Torrent Suite Software to determine the sequences present within the amplicons of these beads.

Two sequencing runs were performed. Sequencing data produced an average read length of 96bp in the first run (FIG. 10) and 94bp in the second run (FIG. 11). The quality measurements of the comparison were 1.76G (AQ 17) and 1.43G (AQ 20) in the first run and 1.48G (AQ 17) and 1.17G (AQ 20) in the second run.

Example 4:

mu.L of beads (forward primer bound) (103X 10) ⁶ mu.L) was mixed with 440. Mu.L of 10mM potassium acetate and 3. Mu.L of 1M Tris (pH 8). The beads were mixed by vortexing and spinning.

mu.L of the DNA fragment with 200bp insert (about 199X 10) was denatured by mixing with 2. Mu.L of NaOH ⁶ Copy), vortexing and spinning, and allowed to stand for 1 minute. The reaction was neutralized by adding 440. Mu.L of 10mM potassium acetate and 3. Mu.L of 1M Tris pH 8. The library comprises a linker linked at one end to a forward primer hybridized to the primer and at the other endOne end is ligated to an insert of a linker hybridized to the reverse primer.

Beads were added to the denatured library. The bead/template ratio was about 10:1 (2000 hundred million beads: 2 hundred million library). Vortex tube and allow to stand at room temperature for 5 minutes (tube 1).

The source of the recombinant enzyme is from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). A list of components in the dehydrated precipitate from Basic kit can be found in example 1 above. Rehydration from the twist amp in 3mL of the rehydration buffer provided by the kit in a 15mL tube (tube 2) ^TM 96 pellets of Basic kit (tube 2).

mu.L of 100. Mu.M reverse primer (non-immobilized primer) and 2. Mu.L of 100. Mu.M biotinylated reverse primer (non-immobilized primer) were added to the washed/prehybridized beads, vortexed (tube 1) and ice-cooled. 1.6mL of an exemplary sieving agent of the present disclosure was added to tube 2, the tube was vortexed for 5 seconds, manually turned/spun for 10 seconds, vortexed for 5 seconds, and placed on ice. mu.L of 280mM magnesium acetate was added to tube 2, the tube was vortexed for 5 seconds, manually flipped/rotated for 10 seconds (vortexed and flipped/rotated 3 times), and placed on ice. 50 μl samples were aliquoted into ice-chilled 96-well plates. The 96-well plates were incubated at 40℃for 60 minutes.

100. Mu.L of 200mM EDTA was added to each well to terminate the reaction. All reactions were pooled and centrifuged at maximum speed for 7 minutes. The supernatant was discarded. The pellet was resuspended in 1mL recovery buffer with 1% SDS, vortexed for 30 seconds, and spun at maximum speed for 6 minutes. After each rotation, the tube was halved by combining the contents of both tubes. The pellet was resuspended in 1mL recovery buffer with 1% SDS, vortexed for 30 seconds, and spun at 1550rpm for 7 minutes.

By interaction with paramagnetic streptavidin beads (MyOne from Dynabeads ^TM Beads) to enrich for the beads that flank copies of the library. During the enrichment step, the ES-washing buffer was replaced with recovery buffer with 0.1% SDS. Finally the beads were resuspended in 1mL of water and reduced to 100 μl. Loading of enriched beads into Ion Torrent Proton I ^TM Ion sensitive coreAnd (3) a sheet.

Sequencing data resulted in an average read length of 144bp (FIG. 12), and the quality measure of the alignment was 4G (AQ 17).

Example 5

At dH ₂ 375x10 was washed in O by vortexing and spinning ⁶ (with forward primer bound). The supernatant (except. About.50. Mu.L) was removed. DNA library (about 75X 10) ⁶ Individual molecules) are added to the washed beads. About 4. Mu.L of reverse primer (non-biotinylated) and 0.4. Mu.L of biotinylated reverse primer were added to the beads. About 0.8. Mu.L of the fusion forward primer was added to the beads. mu.L of magnesium acetate was added to the beads (final concentration 14 mM). Will dH ₂ O was added to the final total volume of the beads to 320 μl.

The source of the recombinant enzyme is from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). A list of components in the dehydrated precipitate from Basic kit can be found in example 1 above. Rehydration from the twist amp in 488 μl of rehydration buffer provided by the kit in a separate tube ^TM 16 pellets of Basic kit. For the 400bp library, 0.25mg/ml of T4 gp32 protein (0.2 mg final concentration) was added, and for the 600bp library, 0.5mg/ml of T4 gp32 protein (0.4 mg final concentration) was added. The vortex tube is rotated and mixed.

The contents of the bead mixture were added to the recombinase tube, vortexed and spun. The bead/recombinase mixture was transferred with pre-chilled oil to a tube and at Ion Torrent OneTouch ^TM Collected on a 10 micron sterlite filter on the device. According to the manufacturer's instructionsBooks create and break emulsions.

By interaction with paramagnetic streptavidin beads (MyOne from Dynabeads ^TM Beads) to enrich the beads in the copy of the library (or omit the enrichment step). The enriched beads were loaded into Ion Torrent Ion sensitive chips and subjected to standard sequencing reactions. A significant portion of the enriched beads are determined to comprise a substantially monoclonal population of amplified polynucleotides, e.g., by Ion Torrent PGM ^TM Observations of detectable sequencing signals from these beads on the sequencer were confirmed. The sequencing signal is analyzed to determine the sequences present within the amplicons of such beads.

Example 6

About 120x10 with annealing buffer ⁶ The beads (with forward primer bound) were washed once. The supernatant (except. About.50. Mu.L) was removed. The washed beads were resuspended in 100. Mu.L of annealing buffer. About 60X10 of DNA library ⁶ Molecules are added to the beads. The final volume was adjusted to 200. Mu.L with annealing buffer. The beads and library were mixed by vortexing and spinning. The bead/library mixture was heated to 95-100 ℃ and held for 3 minutes followed by incubation at 37 ℃ for 5 minutes.

1mL of 10mM potassium acetate was added to the bead/library mixture, followed by vortexing and spinning. The supernatant was discarded. The potassium acetate wash was repeated once. The beads/DNA were resuspended in 120. Mu.L potassium acetate.

The source of the recombinant enzyme is from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). A list of components in the dehydrated precipitate from Basic kit can be found in example 1 above. Rehydrating the samples from the twist Amp in 720. Mu.L of the rehydration buffer provided by the kit in a separate tube ^TM 24 precipitates from Basic kit. An additional 54. Mu.L of dNTP mix (containing 10mM each dNTP) was added to the TwistAmp ^TM And (3) a mixture.

In the third tube, 3. Mu.L of streptavidin (500. Mu.M) was mixed with 12. Mu.L of biotinylated reverse primer (100. Mu.M) and then transferred to the bead/library tube. The recombinase mixture was added to the bead/library tube, followed byThe mixture was then vortexed to mix and ice-cooled. 27. Mu. L T4gp32 protein (15/. Mu.g/. Mu.L) was added to the beads/library tube and vortexed for mixing. 390 μl of the exemplary screening agent was added to the bead/library tube, the tube was inverted and vortexed to mix, ice-cooled for at least 5 minutes. mu.L of magnesium acetate was added to the beads/library tube and the tube was vortexed, spun and ice-cooled for at least 10 seconds. Beads/library tube at 40℃ Wen Yoxiao for 40 min. The reaction was stopped by adding 500. Mu.L EDTA (250 mM). The tube was rotated above 18,000XG for 3 minutes. The supernatant was discarded and the pellet was resuspended in 1mL of TE with 1% SDS. The beads were washed twice in 1mL OneTouch wash solution. The beads were resuspended in 100 μl. The pellet was resuspended by pipetting up and down and by adding 2mL Ion Torrent OneTouch ^TM The washing solution is used for washing. The washing step was repeated once. The pellet was resuspended in 300. Mu.L of melt-off solution and incubated for 5 minutes with shaking.

Example 7

Nucleic acid amplification was performed on Ion sequencing chips. First, a template walking amplification reaction is performed on a chip, followed by a recombinase-mediated amplification reaction.

Preparation of Ion Torrent PGM ^TM Sequencing chip comprising low T _M Is ligated to the bottom of the well at its 5' end. The immobilized primer comprises a polyA (30) sequence.

The double-stranded DNA template comprises a single-stranded terminal protruding sequence having a polyT (30) sequence.

Treatment of Ion Torrent PGM with polymer ^TM The chip was sequenced to create a matrix at the bottom of the well. The capture primer is attached to the substrate.

Pre-washing Ion Torrent PGM with TE-containing buffer ^TM The chip was sequenced once and dried in vacuo.

40 microliters of the solution was mixed and loaded onto the chip. The final concentration of the solution contained: 1 Xisothermal buffer from New England Biolabs, 1.6mM MgSO ₄ 3mM dNTP, 1U/uL Bst polymerase (from New England Biolabs), 0.1nM template and nuclease free water to a volume of 40 uL. The chip was centrifuged for 5 min and incubated at 37℃for 30 min. The chip was dried in vacuo.

Template walking amplification: 40 microliters of template running solution was loaded onto the chip. The final concentration of the template running solution contained: 1 Xisothermal buffer from New England Biolabs, 3.6mM MgSO ₄ 5mM dNTP, 2uM soluble single stranded primer, 6U/uL Bst polymerase (from New England Biolabs) and nuclease free water to 40uL volume. The chips were centrifuged and incubated at 60℃for 30 minutes. The chip was washed once with 1X TE-containing buffer and dried in vacuo.

Recombinase-mediated amplification: the source of the recombinant enzyme of this example is derived from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). A list of components in the dehydrated precipitate from Basic kit can be found in example 1 above. 50 microliters of amplification reaction mixture (containing recombinase) was loaded onto the chip. The amplification reaction mixture comprises: from TwistAmp ^TM One precipitate from Basic kit (from TwistDx, cambridge, great Britain), from TwistAmp ^TM 30uL of rehydration buffer from Basic kit, 2uM of soluble primer hybridized to one adaptor of DNA template and nuclease free water to a total volume of 50 uL. The chip was centrifuged for 2 minutes. 2 microliters of magnesium acetate (280 mM stock) was added to the chip. The chip was centrifuged for 2 minutes and then incubated at 40℃for 1 hour.

The chip was washed successively with 0.5M EDTA (pH 8), TE-containing buffer, 1% SDS, and 2X with the washing solution.

A substantial portion of the wells contained a substantially monoclonal population of amplified polynucleotides were determined using color-coded alignment patterns of the chips.

According to Ion PI ^TM Standard Sequencing reactions were performed according to the manufacturer's instructions in Sequencing 200Kit (User Guide PN MAN 0007491). The sequencing signal was analyzed by Torrent Suite Software to determine the sequences present within the amplicons of these beads. Sequencing data resulted in an average read length of 151 bp.

Example 8

Directly in Ion Torrent PGM in the presence of a recombinase ^TM Nucleic acid amplification is performed under isothermal conditions on a sequencing chip. Placing the polymerized hydrogel on PGM ^TM In the wells of the chip.

The source of the recombinant enzyme of this example is derived from TwitAmp ^TM Basic kit (from TwitDx, cambridge, great Britain). A list of components in the dehydrated precipitate from Basic kit can be found in example 1 above.

The polynucleotide template is denatured using a thermal denaturation method or a recombinase method (as described above), followed by a nucleic acid amplification step.

(A) The thermal denaturation method comprises the following steps: the polynucleotide template library was diluted to a final volume of 60 μl in annealing buffer. Dilution is intended to place about 5 copies of the template into Ion Torrent Proton ^TM Sequencing chip (about 600-650x 10) ⁶ A plurality of holes). The chip was washed once with annealing buffer and placed on a thermal cycler set at 40 ℃. Aliquots of 100. Mu.L of 1:1 annealing buffer to water were mixed and preheated at 95 ℃. The template library was denatured by placing on chip and incubating at 95℃for 2 min. A buffer/water mixture (preheated to 95 ℃) was drawn into the flow cell. The chip was transferred to a 40 ℃ thermocycler and incubated for 5 minutes. The chip was transferred to a laboratory bench (about 25 ℃). The chip was washed with 100. Mu.L of annealing buffer. The chip was placed on a thermal cycler at 4 ℃. The following steps are optional: rehydration from twist amp in 20 μl of water and 30 μl of rehydration buffer provided by the kit ^TM 1 precipitate from Basic kit. The mixture was precipitated by vigorous vortexing to dissolve the precipitate. 50. Mu.L of the pellet mixture was loaded onto a chip. The chip was incubated at room temperature for at least 1 minute to allow the recombinase to bind to the primers preloaded into the wells of the chip.

(B) Recombinant enzyme denaturation method: wash Ion Torrent Proton with 150 μl of annealing/water mixture (1:1 ratio of annealing buffer to water) ^TM Sequencing chip (about 600-650x 10) ⁶ A hole). The chip was placed on a 40 ℃ thermocycler. Rehydration from TwitAmp in 60 μl of rehydration buffer provided by the kit ^TM 2 precipitates from Basic kit. The polynucleotide template library was diluted to a final volume of 50 μl in annealing buffer. Dilution is intended to place about 5 copies of the template into Ion Torrent Proton ^TM Each well of the chip was sequenced. A total volume of diluted template was added to the rehydrated precipitation mixture. The volume was adjusted to 100 μl with water. Vortex the pellet/template to mix and spin. Loading the pellet/template mixture into Ion Torrent Proton ^TM The chip was sequenced (placed on a 40℃thermocycler) and incubated for 20 minutes. The chip was removed from the thermal cycler and allowed to stand at room temperature.

Nucleic acid amplification was performed as follows: all reagents were kept on ice. Rehydration from the TwitAmp in 30. Mu.L of rehydration buffer provided by the kit, 16. Mu.L of nuclease-free water, and 1. Mu.L of 100. Mu.M reverse amplification primer ^TM 1 precipitate from Basic kit. The precipitate was dissolved by vortexing and spinning. Immediately prior to loading into the chip, 3 μl of 280 μΜ magnesium acetate was added to the precipitation mixture. The whole pellet mixture was loaded onto the chip and incubated for 1 hour at 40 ℃.

The amplification reaction was stopped by washing the chip with 0.1M EDTA (pH 8). The chip is washed with the chip washing solution. The chip was washed with 1% SDS. The chip was washed twice with TEX wash solution.

Chips were prepared for sequencing: the chip was washed with the melt-out solution. The chip was washed 3 times with annealing buffer and placed on a 40 ℃ thermocycler. Sequencing primers were prepared in separate tubes: mu.L of 50% annealing buffer was mixed with 50% sequencing primer and then preheated to 95 ℃. A 1:1 mixture of annealing buffer and water was prepared and preheated to 95 ℃. The chip was washed with preheated annealing/water buffer. 80. Mu.L of the preheated primer mixture was loaded into the chip. The chip was incubated at 40℃for 5 minutes. The chip was washed once with annealing/water buffer. In a separate tube, 6 μl of sequencing polymerase was mixed with 57 μl of annealing/water mixture and loaded onto the chip.

Standard sequencing reactions were performed according to the manufacturer's instructions.

Claims

1. A method for nucleic acid amplification comprising:

(a) Partitioning at least two polynucleotides into an array of reaction chambers by introducing a single said polynucleotide into at least two said reaction chambers; and

(b) Forming at least two substantially monoclonal nucleic acid populations by amplifying polynucleotides within the at least two reaction chambers, wherein the at least two reaction chambers are in fluid communication with each other during amplification.

2. The method of claim 1, wherein the at least two polynucleotides have different sequences.

3. The method of claim 1, wherein the amplifying is performed without completely denaturing the at least two polynucleotides during the amplifying.

4. The method of claim 1, wherein the amplifying comprises: contacting the at least two polynucleotides with a single reaction mixture comprising reagents for nucleic acid synthesis.

5. The method of claim 1, wherein the amplifying comprises: contacting the at least two polynucleotides with a recombinase in a reaction chamber.

6. The method of claim 1, wherein the at least two reaction chambers are operably coupled to a sensor.

7. The method of claim 6, wherein the sensor is capable of detecting the presence of nucleotide incorporation byproducts within the reaction chamber.

8. The method of claim 6, wherein the reaction chamber comprises a hydrophilic polymer matrix conformally disposed within the reaction chamber.

9. The method of claim 8, wherein the hydrophilic polymer matrix comprises a hydrogel polymer matrix.

10. The method of claim 8, wherein the hydrophilic polymer matrix is an in situ cured polymer matrix.